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Pregestational population screening of healthy females for copy number variants in DMD gene has raised numerous challenges regarding the interpretation and disclosure of these findings. Our objective was to analyze data from a local dystrophinopathy patient database, in comparison to population screening results. Utilizing the "Little steps" association registry for children with dystrophinopathy, we classified genetic findings (out-of-frame, in-frame, or difficult-to-predict) in 231 DMD and 90 BMD male patients. A comparison was made with a previously published cohort of 162 female carriers identified through population screening. Duplications classified as "difficult to predict" were absent in DMD/BMD patients, as opposed to 45.1% of women analyzed in the scope of population screening (p < 0.0001). While the distribution of deletions did not differ between the groups, significantly higher proportion of duplications initiated at the proximal hot spot in the DMD/BMD cohort (87.1%), vs. only 11.7% in women analyzed through population screening (p = 0.0038). Notably, duplications initiating in the dp427c promoter area were noted only in the latter cohort (n = 62). Local databases of dystrophinopathy patients can facilitate analysis and reporting of pregestational female population screening results. These conclusions facilitate future introductions of population screening genetic tests for diseases with variable presentation. [ABSTRACT FROM AUTHOR]

Species identification of Listeria isolates remained a tedious process still based on culturing methods that, in recent years, have led to the description of many species that are not even part of the genus Listeria. It is advisable to provide new precise techniques since this taxon includes two pathogens that are usually transmitted through the food chain, Listeria monocytogenes and L. ivanovii. The approach, so-called multilocus phylogenetic analysis (MLPA) that uses several concatenated housekeeping gene sequences, provides accurate and affordable classification frameworks to easily identify Listeria species by simple Sanger sequencing. Fragments of seven housekeeping genes (gyrA, cpn60, parE, recA, rpoB, atpA, and gyrB) from 218 strains of all Listeria species currently described were used to build an MLPA of the concatenated sequence, a total of 4375 bp. All isolates subjected to identification were clustered within the species of Listeria sensu stricto, L. monocytogenes, L. innocua, and L. welshimeri, and some reference strains were reclassified as L. ivanovii and L. seeligeri. Housekeeping-gene sequencing has been demonstrated to represent a pragmatic tool that can be firmly considered in food control.

α-thalassemia (α-thal) is a genetic disorder characterized by a decreased synthesis of α-globin chains. A deletion mutation most often causes it in one or more α-globin chains. No comprehensive characterization studies have been conducted on α-thal patients in the Saudi population. Therefore, this research aims to identify the spectrum of genetic mutations responsible for α-thal in our region. Individuals with microcytic, hypochromic red blood cells and normal hemoglobin (Hb) A2 were enrolled. Sixty samples of individuals suspected of α-thal were selected for further genetic analysis. Multiplex ligation-dependent probe amplification assay was used to detect deletion mutations in α-globin genes. Among all samples tested, the -α3.7 deletion mutation was detected in 57 (95%) cases, whereas no mutation was detected in the remaining 3 (5%). In addition, 9 (15%) individuals were heterozygous for -α3.7, while -α3.7 homozygosity was found in 85% of the analyzed cases. The hematological characteristics of α3.7 subjects were significantly lower than the control group in the mean of Hb, hematocrit, mean corpuscular volume, mean corpuscular Hb, and mean corpuscular Hb concentration (p<0.001). These results highlight the importance of α-thal diagnosis and investigation in Saudi Arabia's pre-marital screening program for microcytic hypochromic individuals. Thus, it contributes to reducing the spread of genetic diseases. [ABSTRACT FROM AUTHOR]

Species identification of Listeria isolates remained a tedious process still based on culturing methods that, in recent years, have led to the description of many species that are not even part of the genus Listeria. It is advisable to provide new precise techniques since this taxon includes two pathogens that are usually transmitted through the food chain, Listeria monocytogenes and L. ivanovii. The approach, so-called multilocus phylogenetic analysis (MLPA) that uses several concatenated housekeeping gene sequences, provides accurate and affordable classification frameworks to easily identify Listeria species by simple Sanger sequencing. Fragments of seven housekeeping genes (gyrA, cpn60, parE, recA, rpoB, atpA, and gyrB) from 218 strains of all Listeria species currently described were used to build an MLPA of the concatenated sequence, a total of 4375 bp. All isolates subjected to identification were clustered within the species of Listeria sensu stricto, L. monocytogenes, L. innocua, and L. welshimeri, and some reference strains were reclassified as L. ivanovii and L. seeligeri. Housekeeping-gene sequencing has been demonstrated to represent a pragmatic tool that can be firmly considered in food control. [ABSTRACT FROM AUTHOR]

Spinal Muscular Atrophy (SMA) is a neuromuscular disease due to degeneration of the anterior horn cells of the spinal cord. The estimated incidence of SMA is 1:6,000-1:10,000. The complete deletion of exon 7 of the SMN1 gene is the hallmark of 95-98% of SMA patients in most population. The first line of investigation for a child or young adult patient suspected to have SMA should be Multiplex ligation-dependent probe amplification (MLPA) testing for homozygous deletion of exons 7 and exon 8 in the SMN1 gene. In this paper, we report the results of SMN 1 exon 7 deletion tests in children who attended the Genetic clinic of a tertiary care hospital in Kerala with one or more of the symptoms especially floppy infants, hypotonia, muscle weakness, tongue fasiculations etc. SMN1 exon 7 and exon 8 deletion was confirmed in 58% cases (19) of the total 33 hypotonia patients. SMA Type I, Type II and Type III were 68.4% (13), 21% (4) and 10.5% (2) respectively among the SMA positive cases. Carrier testing of the non-consanguineous parents showed that all parents were heterozygous carriers. Until 2016, the treatment for this disease was supportive only. Recently Nusinersen, Zolgensma and Risdiplam have become available for SMA patients. The carrier testing in parents with previous SMA child history is essential for the implementation of prenatal diagnosis of this disorder in future pregnancies. The paper emphasizes the importance of this rare neuromuscular disease. [ABSTRACT FROM AUTHOR]

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a heterogeneous group of symptoms, including characteristic cafe-au-lait macules, axillary or inguinal freckling, Lisch nodules, as well as skeletal deformation, scoliosis, mental retardation, and tumors of the nervous system. Klinefelter syndrome (KS) is a gonadal dysgenesis, with symptoms in males, including an extra X chromosome, leading to tall stature, hypogonadism, and infertility. Although the co-occurrence of NF1 and KS is a rare finding, this report describes this unique entity detected in an eight-year-old boy with numerous hyperpigmentation spots, a multitude of skin and subcutaneous nodules, seizures, arterial stenosis, and mild gynecomastia. Whole-exome sequencing (WES) was conducted along with copy number analysis for the proband and his parents. Multiplex Ligation-dependent Probe Amplification (MLPA) is used to validate the copy number variations detected by next-generation sequencing (NGS). The results showed a pathogenic heterozygous mutation (c.246_247del, p.Gln83Valfs*23) in a human neurofibromin 1 gene (NF1), detected in the proband and his father, whilst the genetic analysis performed by the karyotype revealed a copy gain of the X chromosome (47, XXY) leading to KS. This rare occurrence of NF1 with co-occurrence of KS may raise some concerns and difficulties in the clinical management of this case, particularly Testosterone hormone replacement therapy and the potential risks of malignancies. Therefore, clinicians may ask for KS genetic tests in male patients with NF1 who have symptoms of gynaecomasia or infertility, and closely monitor for potential malignancies and other complications. Compellingly, this case emphasizes the importance of advanced genetic analysis in providing genetic tools for diagnosing and managing individuals with rare and complex syndromes with overlapping clinical features. Early detection and comprehensive clinical interventions are the key cornerstones to improving patient outcomes.

IntroductionGenetic obesity is considered a rare disease, affecting up to 10% of patients with severe early-onset obesity. Over the past years, significant advances have been made; however, the majority of patients are misdiagnosed with polygenic obesity. Thus, this study aimed to identify deleterious copy number variations (CNVs) linked to obesity and explore the clinical phenotypes.MethodThe sample comprised 195 adults with severe obesity (BMI≥35kg/m2) who developed this phenotype during childhood or adolescence. We investigated the CNV using Multiplex Ligation-dependent Probe Amplification [MLPA] and real-time PCR. Chromosomal microarray analysis was used to assess the extent of genomic alterations.ResultsOne patient showed a ~206 kb deletion in the distal of the 16p11.2 region, encompassing twelve genes. The gene linked to the development of severe obesity was SH2B1. This alteration was found in a male patient with metabolic syndrome (MS), and hypertension. Two patients exhibited a large deletion in the proximal of the 16p11.2 region. One patient showed a ~534 kb deletion without twenty-nine genes. This female patient had hypertension and bronchitis. The other patient presented a ~598 kb deletion of the proximal 16p11.2 region, including thirty-two genes. This female patient exhibited MS, and moderate binge-eating disorder.ConclusionOur study showed three genomic deletions at the 16p11.2 region in patients with severe obesity from Brazil. These results support the clinical utility of genetic testing to identify patients with the genetic form of obesity who may benefit from specific medical treatment, family genetic counseling, and targeted therapeutic intervention.

Waardenburg syndrome (WS) is a common genetic cause of syndromic hearing loss, accounting for 2–5% of congenital cases. It is characterized by hearing impairment and pigmentation abnormalities in the skin, hair, and eyes. Seven genes are associated with WS: PAX3, MITF, EDNRB, EDN3, SOX10, KITLG, and SNAI2. This study investigates the genetic causes of WS in three familial cases. Whole-exome sequencing (WES) was performed to identify single nucleotide variants (SNVs). Copy number variants (CNVs) were analyzed from the WES raw data and through multiplex ligation-dependent probe amplification (MLPA). The study identified one pathogenic SNV and two novel CNVs, corresponding to type I and type II WS patterns in the three families. The SNV, a nonsense variant (c.1198C>T p.Arg400*), was found in MITF and segregated in the affected father. The two CNVs were a deletion of exon 5 in PAX3 in a family with two affected members and a large novel deletion comprising seven genes, including SOX10, in a family with three affected members. These findings confirmed a WS diagnosis through genetic testing. The study emphasizes the importance of integrating multiple genetic testing approaches for accurate and reliable diagnosis, highlighting their role in improving patient management and providing tailored genetic counseling. [ABSTRACT FROM AUTHOR]

The 22q11.2 region is highly susceptible to genomic rearrangements leading to multiple genomic disorders, including 22q11.2 microdeletion syndrome (22q11.2 DS) (MIM# 188400), 22q11.2 microduplication syndrome (MIM# 608363), supernumerary der(22)t(11;22) syndrome (also known as Emanuel Syndrome; MIM# 609029), and Cat Eye Syndrome (MIM# 115470). In this study, we present data on causes of mortality, average age of death, and the existing associated risk factors in patients with 22q11.2 rearrangements. Our cohort included 223 patients (120 males and 103 females) with confirmed diagnoses of 22q11.2 rearrangements diagnosed through molecular techniques (FISH, MLPA, and CMA). Relatives from patients who have been molecularly confirmed with 22q11.2 rearrangements have also been added to the study, regardless of the presence or absence of symptoms. Of these 223 individuals, 21 (9.4%) died. Deceased patients' rearrangements include 19 microdeletions, 1 microduplication, and 1 patient with a marker chromosome. The median age of death was 3 months and 18 days (ranging from 3 days to 34 years). There were 17 patients who died at pediatric age (80.95%), 3 died at adult age (14.28%), and for 1 of whom, the age of death is unknown (4.76%). Eighteen patients were White Mediterranean (European non-Finnish) (85.71%) whereas three were Amerindian (South American) (14.28%). Mortality from cardiac causes accounted for 71.42%. The second most frequent cause of death was sepsis in two patients (9.52%). One patient died from respiratory failure (4.76%) and one from renal failure (4.76%). Information regarding the cause of death was not available in two patients (9.52%). Most patients who died were diagnosed within the first week of life, the majority on the first day. This study adds additional information on mortality in one of the largest cohorts of patients with 22q11.2 rearrangements in more than 30 years of follow-up. [ABSTRACT FROM AUTHOR]

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Background: Fanconi anemia is a genetically heterogeneous recessive disorder distinguished by cytogenetic instability, hypersensitivity to DNA crosslinking agents, increased chromosomal breakage, and disturbed DNA repair. To date, Fanconi anemia complementation group (FANC) includes 23 FANC genes identified of which, FANCA gene is the most commonly mutated. The mutation spectrum of the FANCA gene is highly heterogeneous with large intragenic deletions due to Alu elements-mediated recombination.The study aimed to identify different deletion mutations on FANCA gene in Egyptian Fanconi anemia patients by multiplex ligation-dependent probe amplification (MLPA) technique to define the spectrum of FA molecular pathology as a step for disease control. The study included 80 FA patients (36 females and 44 males) whose ages ranged from 4 months to 17 years descending from unrelated consanguineous families referred to the Hereditary Blood Disorders Clinic, National Research Centre (NRC), Egypt. Patients were diagnosed with classical clinical presentation of FA and were confirmed by chromosomal breakage using Diepoxybutane (DEB). Results: The common clinical presentation in our FA patients were the presence of café au lait spots with hyperpigmentation in 65/80 (81%) followed by skeletal defects in 40/80 (50%). MLPA revealed a total of five different intragenic homozygous deletions of FANCA gene in 16 /80 (20%) patients, among them two deletion patterns were novel. Conclusion: Molecular analysis using MLPA could detect pathogenic mutations in 20% of FA patients, our study generated considerable data on causative mutations that was used for genetic counseling and prenatal diagnosis.

Duchenne muscular dystrophy (DMD/BMD) is the most common type of muscular dystrophy, together with Becker muscular dystrophy represent more than half of all cases. DMD is a single-gene, X-linked recessive disorder that predominantly affects boys, causing progressive muscle deterioration and eventually leading to fatal cardiopulmonary complications. This study aimed to implement a cost-effective molecular diagnostic method using the SALSA MLPA Kit (probe mixes 034 and 035) to screen a large group of Egyptian DMD patients. The study included 1250 clinically diagnosed DMD males, following complete family history, pedigree analyses and an accurate clinical examination and laboratory investigations mainly considering high levels of creatine phosphokinase (>2000 U/L). We also analysed the carrier status of 100 mothers of 100 probands to gauge the inherited mutation through their patients with familial disease. The negative results of MLPA were further analysed with NGS for ten patients and the results were validated for novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster.Results SALSA MLPA analysis confirmed the diagnoses in 733/1250 (58.6 %) DMD patients and the remaining of 517/1250 (41.4%) were negative. DMD patients having large deletions were 632/1250 (50.6%) while duplications occurred in 101/1250 (8%). The most common single exon deletion was 45 (50/632, 7.9%). In addition, 163 different deletion and duplication patterns were characterized among positive MLPA analyses. 30% of our studied cohort exhibited new patterns of rearragements in addition to seven cases of double deletion and duplication rearrangements identified, within nine patients. Using NGS, for small mutations detection, revealed six novel and three previously reported mutations among screened ten patients.In conclusion, our findings expand the spectrum of known DMD mutations by offering an effective diagnostic method and identifying novel point mutations through NGS analysis. We recommend using NGS to uncover uncharacterized mutations in patients who test negative with MLPA, which could contribute to the treatment of DMD.

Glioblastoma is the commonest primary malignant brain tumor, with a very poor prognosis and short overall survival. It is characterized by its high intra- and intertumoral heterogeneity, in terms of both the level of single-nucleotide variants, copy number alterations, and aneuploidy. Therefore, routine diagnosis can be challenging in some cases. We present a complicated case of glioblastoma, which was characterized with five cytogenomic methods: interphase fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, comparative genomic hybridization array and single-nucleotide polymorphism, targeted gene panel, and whole-genome sequencing. These cytogenomic methods revealed classical findings associated with glioblastoma, such as a lack of IDH and TERT mutations, gain of chromosome 7, and loss of chromosome 10. At least three pathological clones were identified, including one with whole-genome duplication, and one with loss of 1p and suspected loss of 19q. Deletion and mutation of the TP53 gene were detected with numerous breakends on 17p and 20q. Based on these findings, we recommend a combined approach to the diagnosis of glioblastoma involving the detection of copy number alterations, mutations, and aneuploidy. The choice of the best combination of methods is based on cost, time required, staff expertise, and laboratory equipment. This integrated strategy could contribute directly to tangible improvements in the diagnosis, prognosis, and prediction of the therapeutic responses of patients with brain tumors.

Abstract 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

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• About 50 % of patients with BCR-ABL1 negative had CNA-PR. • IKZF1 and CDKN2A deletion correlated with a positive MFC-MRD value on day 15. • The presence of more than one deletion simultaneously correlated with a significantly higher induc-tion 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. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Short stature homeobox (SHOX) haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis. The worldwide prevalence of SHOX variations in ISS varies from 2.5% to 15.0%. The aim of this study was to assess the implication of SHOX variation in ISS in North Indians and compare this with other cases of SHOX variations from Asian population. METHODS: SHOX gene analysis was carried out by multiplex ligation-dependent probe amplification followed by Sanger sequencing in 54 patients with variable phenotypes. Comparison with other reports in a meta-analysis comprising the current study and 11 previous studies (n=979) was performed. RESULTS: SHOX analysis resulted in 12.9% positivity (7.4% deletions and 5.5% duplications). SHOX association was seen significantly related to gender, with predominance in females (p=0.047). Short arms and forearms were the only significantly associated trait seen in 51.9% of children. The overall prevalence of SHOX variation was 15.2% in Asians with ISS. No significant difference was found in geographical region-specific analysis. DISCUSSION AND CONCLUSION: This study summarises findings from the last decade and provides an updated picture of the prevalence of SHOX variations in Asians, emphasizing their potential as therapeutic targets in ISS patients. Further high quality, large investigations including functional validation is warranted to validate this association.

Chromosomal abnormalities are the most common causes of early pregnancy losses (EPLs). In this study, we aimed to evaluate the incidence and spectrum of chromosomal abnormalities in EPLs and correlate them with different clinical characteristics. We performed Quantitative Fluorescent PCR (QF-PCR), followed by subtelomeric Multiplex Ligation Probe Amplification (MLPA) analysis to detect chromosomal abnormalities in 900 products of conceptions (POCs) from EPLs collected over a period of 10 years.

Abstract 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.

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.

Objective: Short stature homeobox (SHOX) haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis. The worldwide prevalence of SHOX variations in ISS varies from 2.5% to 15.0%. The aim of this study was to assess the implication of SHOX variation in ISS in North Indians and compare this with other cases of SHOX variations from Asian population. Methods: SHOX gene analysis was carried out by multiplex ligation-dependent probe amplification followed by Sanger sequencing in 54 patients with variable phenotypes. Comparison with other reports in a meta-analysis comprising the current study and 11 previous studies (n=979) was performed. Results: SHOX analysis resulted in 12.9% positivity (7.4% deletions and 5.5% duplications). SHOX association was seen significantly related to gender, with predominance in females (p=0.047). Short arms and forearms were the only significantly associated trait seen in 51.9% of children. The overall prevalence of SHOX variation was 15.2% in Asians with ISS. No significant difference was found in geographical region-specific analysis. Conclusion: This study summarises findings from the last decade and provides an updated picture of the prevalence of SHOX variations in Asians, emphasizing their potential as therapeutic targets in ISS patients. Further high quality, large investigations including functional validation is warranted to validate this association. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A pregnant woman living in Fujian Province, southeastern China, presented due to a risk of having a baby with β-thalassemia major, during her second pregnancy, since she and her husband were suspected as β-thalassemia carriers and their affected daughter was a transfusion-dependent patient. Using the common α-thalassemia and β-thalassemia genotypes test, the pregnant woman was diagnosed as a β-thalassemia carrier with βIVS−2 − 654 (C→T)/βN 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 β0-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 β-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 βIVS−2 − 654 (C→T)/βFilipino 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 β0-deletion in a Chinese family, from Fujian area, for the first time, during prenatal screening. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a rare, recessively inherited neurodegenerative disorder caused by the presence of pathogenic variants in the SMN gene. As it is the leading inherited cause of infant mortality, identification of SMN gene pathogenic variant carriers is important for diagnostic purposes with effective genetic counseling. Multiple ligation probe analysis (MLPA), a probe-based method, is considered as the gold standard for SMA carrier analysis. However, MLPA might give false-negative results in cases with variations in the probe-binding regions. Here, we present a case born to consanguineous SMA carrier parents. Prenatal diagnosis with MLPA failed to detect the compound heterozygous mutant state of the proband and she was born unfortunately with SMA phenotype. Further analysis with a real-time polymerase chain reaction kit was able to detect the compound heterozygous state of the patient and was confirmed with targeted next-generation sequencing technology.

β-thalassemia is a common inherited hemolytic disorder caused by mutations in the HBB gene. Genetic analysis of 2 new beta-thalassemia patients with deletion mutations in the HBB gene and their family members. Their clinical presentation and blood phenotypic tests were analyzed. We detected the approximate degree of deletion of these two new HBB gene deletion mutants and analyzed their specific deletion locations by multiplex ligation-dependent probe amplification (MLPA), reverse breakpoint polymerase chain reaction (GAP-PCR), and sanger DNA sequencing. Two new deletion mutants of the HBB gene were identified. First, a 49% decrease in the expression of the third exon of the HBB gene was detected by MLPA testing, and then proband 1 and her mother were found to have HBB: exon3del and proband 2 and her mother to have HBB: c.−81A > C by GAP-PCR and sanger sequencing. When the blood phenotype and clinical manifestations do not match the genotype, the presence of new mutants should be considered, and attention should be paid to further testing to avoid missing the diagnosis, which can help in clinical diagnosis and treatment, prenatal diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

Chromosomal abnormalities are the most common causes of early pregnancy losses (EPLs). In this study, we aimed to evaluate the incidence and spectrum of chromosomal abnormalities in EPLs and correlate them with different clinical characteristics. We performed Quantitative Fluorescent PCR (QF-PCR), followed by subtelomeric Multiplex Ligation Probe Amplification (MLPA) analysis to detect chromosomal abnormalities in 900 products of conceptions (POCs) from EPLs collected over a period of 10 years. Chromosomal abnormalities were present in 56.25% of uncontaminated EPLs, with significantly higher incidence in women ≥36 years (71.37%, p<0.0001) in comparison to women ≤30 years of age (43.40%). Trisomies were also more common in women ≥36 years (79.68%, p<0.0001) than in those ≤30 years of age (48.70%). In contrast, triploidy and monosomies were more prevalent in women ≤30 years of age (26.09%, p<0.0001 and 16.52%, p=0.0066 respectively) than in women ≥36 years of age (6.42% and 6.42% respectively). Trisomy 16 was more common in women ≤30 (39.29%, p=0.0009) than in those ≥36 years of age (16.78%), while trisomy 22 was predominant among women ≥36 (23.49%, p=0.013), and was not present in the group of women ≤30 years of age. The frequency of chromosomal abnormalities in POCs from women with sporadic (61.19%) was higher than in those with recurrent EPLs (55.21%). This difference, however, was not statistically significant (p=0.164). Although some differences in the chromosomal aneuploidy rates among women with different ABO blood groups, as well as among 6–8 and 9–11 gestational week EPLs were observed, further larger studies are required to confirm these findings. In conclusion, our study enriches the knowledge about chromosomal abnormalities as a cause of EPLs and confirms the higher incidence of foetal chromosomal abnormalities in EPLs in women of older reproductive age. Furthermore, it shows that using QF-PCR and MLPA methodologies, a high detection rate of chromosomal abnormalities in EPLs can be reached. [ABSTRACT FROM AUTHOR]

Background: Retinoblastoma (RB), an intraocular malignancy commonly diagnosed in children, is mostly caused by inactivating mutations of both alleles of the RB1 gene. Early genetic screening for RB1 gene mutations would greatly improve treatment outcomes and patient management. Methods: In this study, both somatic and germline mutations were detected in blood and tumour samples of 42 RB patients using direct sequencing and multiplex ligation‐dependent probe amplification. Results: In total, 34 different mutations were found in 36 patients, including 1 SNP, 4 large deletions, 5 splicing sites, 1 missense, 7 frameshifts and 17 nonsense mutations. There were five novel mutations and one unreported which have not been found in large databases such as Leiden Open Variation Database (LOVD) and ClinVar. Conclusion: A higher rate of RB patients carrying heterozygous germline mutation and highly prevalent with pathogenic truncated mutation, hence, early detection of RB is essential for vision salvation and genetic counselling. [ABSTRACT FROM AUTHOR]

We present the case of a male patient who was ultimately diagnosed with Becker muscular dystrophy (BMD; MIM# 300376) after the onset of muscle weakness in his teens progressively led to significant walking difficulties in his twenties. A genetic diagnosis was pursued but initial investigation revealed no aberrations in the dystrophin gene (DMD), although immunohistochemistry and Western blot analysis suggested the diagnosis of dystrophinopathy. Eventually, after more than 10 years, an RNA analysis captured abnormal splicing where 154 nucleotides from intron 43 were inserted between exon 43 and 44 resulting in a frameshift and a premature stop codon. Normal splicing of the DMD gene was also observed. Additionally, a novel variant c.6291–13537A>G in DMD was confirmed in the genomic DNA of the patient. The predicted function of the variant aligns with the mRNA results. To conclude, we here demonstrate that mRNA analysis can guide the diagnosis of non-coding genetic variants in DMD. [ABSTRACT FROM AUTHOR]

X-linked recessive ichthyosis (XLI) is clinically characterized by dark brown, widespread dryness with polygonal scales. We describe the identification of STS and PUDP deletions using targeted panel sequencing combined with copy-number variation (CNV) analysis in XLI. A 9-month-old infant was admitted for genetic counseling. Since the second day after birth, the infant's skin tended to be dry and polygonal scales had accumulated over the abdomen and upper extremities. The infant's maternal uncle and brother (who had also exhibited similar skin symptoms from birth) presented with polygonal scales on their trunks. CNV analysis revealed a hemizygous deletion spanning 719.3 Kb on chromosome Xp22 (chrX:7,108,996–7,828,312), which included a segment of the STS gene and exhibited a Z ratio of −2 in the proband. Multiplex ligation-dependent probe amplification (MLPA) confirmed this interstitial Xp22.31 deletion. Our report underscores the importance of implementing CNV screening techniques, including sequencing data analysis and gene dosage assays such as MLPA, to detect substantial deletions that encompass the STS gene region of Xq22 in individuals suspected of having XLI. [ABSTRACT FROM AUTHOR]

Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

In humans particularly in children, adenovirus is one of the most common viruses that cause respiratory illnesses. Knowing how to detect adenovirus proficiently and rapidly will help reinforce surveillance of adenovirus infections, detect epidemic situations in real-time, and understand the trend of virus epidemics, which will allow effective actions to be taken quickly. The rapid detection of antiviral antibodies or viral antigens in clinical samples can be achieved by molecular diagnostic techniques like PCR, Real-Time PCR, LAMP, mPCR-RLB, PCR-ELISA, Tem-PCR, Gene Chip, and so on. Some of the molecular diagnostic methods are relatively economical, exceedingly sensitive and explicit. There are several commercially accessible molecular diagnostic techniques that enable their use in clinical laboratories all over the world. In this review, the principles, characteristics, and applications of molecular biology surveillance methods commonly used in labs and clinics for the detection of human adenoviruses are examined and highlighted.

Abstract Spinal muscular atrophy (SMA) is considered one of the most common autosomal recessive disorders, with an estimated incidence of 1 in 10,000 live births. Testing for SMA has been recommended for inclusion in neonatal screening (NBS) panels since there are several therapies available and there is evidence of greater efficacy when introduced in the pre/early symptomatic phases. In Brazil, the National Neonatal Screening Program tests for six diseases, with a new law issued in 2021 stating that it should incorporate more diseases, including SMA. In the present study, dried blood spot (DBS) samples collected by the Reference Services of Neonatal Screening of RS and SP, to perform the conventional test were also screened for SMA, using real-time PCR, with SALSA MC002 technique. A total of 40,000 samples were analyzed, enabling the identification of four positive cases of SMA, that were confirmed by MLPA. Considering our sampling, Brazil seems to have an incidence comparable to the described in other regions. This work demonstrated that the use of the MC002 technique in samples routinely collected for the conventional NBS program is suitable to screen for SMA in our conditions and can be included in the expansion of the neonatal screening programs.

Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Introduction. SMA is a life-threatening autosomal recessive, neuromuscular disorder that affects the nerve cells controlling the muscles. SMA is caused by mutations in the SMN1 gene and results in neuronal degeneration and muscular atrophy Early detection of SMA through newborn screening (NBS) is essential for preventing irreversible damage by selecting appropriate treatment and ensuring adequate follow-up. Aim: to implement genetic screening for spinal muscular atrophy (SMA) as a tool in the early diagnosis of spinal amyotrophy. Materials and methods. In the Human Molecular Genetics Laboratory at the Mother and Child Institute (IM&C), an algorithm has been implemented for genetic screening of SMA-associated mutations. This includes the collection of blood samples on filter paper cards from newborns and their analysis using molecular genetic methods such as real-time PCR and MLPA. Results. In order to implement the proposed algorithm, the necessary molecular genetic methods were employed, including real-time PCR method, for which specific probes and matrices were designed. The analysis set was validated using 10 samples with SMN1 exon 7 deletion and 10 samples without SMN1 exon 7 deletion. Furthermore, the MLPA method was implemented with the aim of confirming and assessing the copy number of SMN1/ SMN2 genes. After obtaining approval for the research design and protocol for this initiative, informed consent and acceptance forms were developed to confirm the participants' willingness to participate in the study, which were approved by the Research Ethics Committee of the USMF "N. Testemițanu". The minimum number of study participants was calculated using the EpiInfo 7.2.2.6 program, specifically the "StatCalc-Sample Size and Power" module, with a minimum sample size calculated to be 172 participants. Subsequently, 250 blood spot samples were collected from newborns, with 75 samples already undergoing genetic screening. Following the analysis of the results, no sample was declared to have a suspected SMA status. Conclusions. The implemented algorithm for newborn screening for SMA detects newborns affected by SMA caused by a homozygous deletion of SMN1 exon 7. SMA due to compound heterozygous mutations (approximately 5% of SMA individuals) is not detected by this test. Newborn screening can identify patients affected by SMA before the onset of symptoms and provides an opportunity for early therapeutic intervention. The implementation of genetic screening for SMA within Institute of Mother and Child offers the chance to assess its feasibility as an early diagnostic tool for SMA and its benefits for the healthcare system of the Republic of Moldova. [ABSTRACT FROM AUTHOR]

Background: Prader-Willi Syndrome (PWS) is a multisystemic disorder characterized by dysmorphic facies, hypotonia, developmental delay, cognitive impairment, hypogonadism, and obesity. It is caused by the absence of expression of paternally derived genes on chromosome 15. Here, we report the diagnostic journey of a case with severe neonatal hypotonia. Case Report: A neonatal patient was referred for the prediagnosis of spinal muscular atrophy (SMA). During the SMA Multiplex Ligation-dependent Probe Amplification (MLPA) analysis, a diminished signal of a reference probe on the 15q11.2 was revealed. Later, it was confirmed that she had a deletion confined to 15q11.2-q13.1, with a methylation pattern compatible with PWS. Conclusion: Since hypotonia might be the only finding in newborns with PWS, this case was presented to emphasize the importance of a comprehensive approach to such patients. [ABSTRACT FROM AUTHOR]

Background/Aim: Germline copy number variation (CNV) is a type of genetic variant that predisposes significantly to inherited cancers. Today, next-generation sequencing (NGS) technologies have contributed to multi gene panel analysis in clinical practice. Materials and Methods: A total of 2,163 patients were screened for cancer susceptibility, using a solution-based capture method. A panel of 52 genes was used for targeted NGS. The capture-based approach enables computational analysis of CNVs from NGS data. We studied the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and of the non-commercial tool panelcn.MOPS. Additionally, we tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA). Results: Pathogenic/likely pathogenic variants (P/LP) were identified in 464 samples (21.5%). CNV accounts for 10.8% (50/464) of pathogenic variants, referring to deletion/duplication of one or more exons of a gene. In patients with breast and ovarian cancer, CNVs accounted for 10.2% and 6.8% of pathogenic variants, respectively. In colorectal cancer patients, CNV accounted for 28.6% of pathogenic/likely pathogenic variants. Conclusion: In silico CNV detection tools provide a viable and cost-effective method to identify CNVs from NGS experiments. CNVs constitute a substantial percentage of P/LP variants, since they represent up to one of every ten P/LP findings identified by NGS multigene analysis; therefore, their evaluation is highly recommended to improve the diagnostic yield of hereditary cancer analysis. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic leukemia (CLL) is known for its wide-ranging clinical and genetic diversity. The study aimed to assess the associations between copy number variations (CNVs) and various biological and clinical features, as well as the survival rates of CLL patients and to evaluate the effectiveness of the multiplex ligation-dependent probe amplification (MLPA) technique in CLL patients.DNA was extracted from 110 patients, and MLPA was performed. Mutations in NOTCH1, SF3B1, and MYD88 were also analyzed. A total of 52 patients showed at least one CNV, 26 had at least one somatic mutation, and 10 presented both, CNVs, and somatic mutations. The most commonly identified CNVs were del(114.3), del(11q22.3), and dup(12q23.2). Other CNVs identified included del(17p13.1), del(14q32.33), dup(10q23.31), and del(19p13.2). One patient was identified with concomitant trisomy 12, 13, and 19. NOTCH1 and SF3B1 mutations were found in 13 patients each, either alone or in combination with other mutations or CNVs, while MYD88 mutation was identified in one patient. Forty-two patients had normal results. Associations between the investigated CNVs and gene mutations and patients' overall survival were found. The presence of NOTCH1 and SF3B1 mutations or the combination of NOTCH1 mutation and CNVs significantly influenced the survival of patients with CLL. Both mutations are frequently associated with different CNVs. Del(13q) is associated with the longest survival rate, while the shortest survival is found in patients with del(17p). Even if MLPA has constraints, it may be used as the primary routine analysis in patients with CLL. [ABSTRACT FROM AUTHOR]

Background: Lonicera Japonicae Flos (LJF) and Lonicerae Flos (LF), derived from different species of Lonicera, are two different types of traditional Chinese medicine (TCM) according to their differences in composition and effectiveness. Because of the high price, good effectiveness of the medicinal, and functional values, LJF is often adulterated by LF in the market. Therefore, two single nucleotide polymorphism‐based genotyping methods, multiplex ligation‐dependent probe amplification high‐resolution melting (MLPA‐HRM) and RNase H2‐dependant PCR (rhPCR)‐HRM, were established for the identification of LF and LJF. Results: Highly specific MLPA probes and rhPCR‐primers were designed based on the trnL‐trnF regions. The melting temperature (Tm) of amplicons was derived from the peak of the HRM curve, and the differences in Tm values were used to identify LJF and LF. The two methods have both shown good specificity for identifying LJF and LF without cross‐reaction, and have high sensitivity with the detection limits of 0.1 ng DNA template. For mixed samples, MLPA‐HRM could detect LJF mixed with 10% LF while rhPCR‐HRM could detect 5% LF in mixed samples. The MLPA‐HRM and rhPCR‐HRM were performed on DNA extracted from 54 samples of LJF and LF randomly selected from the medicinal material market, all samples were successfully identified and further verified with DNA barcoding. Conclusion: MLPA‐HRM and rhPCR‐HRM methods developed in our study not only provided fast, simple, sensitive, and practical identification of LJF and LF, but also provided technical references for the adulteration identification of other herb‐medicines, they are of great significance to the quality control and safe use of plant medicinal materials. [ABSTRACT FROM AUTHOR]

Abstract Background Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease representing the most prevalent monogenic cause of infant mortality. It results from the loss of SMN1 gene, but retention of its paralog SMN2 whose copy number can modulate the disease severity and guide the therapeutic regimen. Methods For SMA molecular analysis, 236 unrelated Egyptian patients were enrolled at our institution. The Multiplex ligation-dependent probe amplification analysis (MLPA) was applied to investigate the main genetic defect in the enrolled patients (SMN1 loss) and to determine a possible genotype–phenotype correlation between the copy number of other genes in the SMN locus (5q13.2) and disease severity in Egyptian patients with SMA. A small cohort of healthy subjects (n = 57) was also included to investigate the possible differences in the distributions of SMN2 and NAIP genes between patients and healthy individuals. Results Disease diagnosis was confirmed in only 148 patients (62.7%) highlighting the clinical overlapping of the disease and emphasizing the importance of molecular diagnosis. In patients with homozygous SMN1 loss, the disease was mediated by gene deletion and conversion in 135 (91.2%) and 13 (8.8%) patients, respectively. In the study cohort, SMN2 and NAIP copy numbers were inversely correlated with disease severity. However, no significant association was detected between GTF2H2A and SERF1B copy numbers and patient phenotype. Significant differences were demonstrated in the copy numbers of SMN2 and NAIP between SMA patients and healthy subjects. Conclusion Molecular analysis of SMA is essential for disease diagnosis. Consistent with previous studies on other populations, there is a close relationship between SMN2 and NAIP copy numbers and clinical phenotype. Additionally, potential differences in these two genes distributions are existing between patients and healthy subjects. National program for carrier screening should be established as a preventive disease strategy. On the other hand, neonatal testing would provide accurate estimation for disease incidence.

Abstract Inherited neurotransmitter diseases are a subset of rare neurometabolic disorders characterized by hereditary deficiencies in neurotransmitter metabolism or transport. Non-ketotic hyperglycinaemia (NKH), called glycine encephalopathy, is an autosomal recessive glycine metabolism disorder characterized by an abnormal accumulation of glycine in all bodily tissues, including the CNS. The SLC6A9 gene, which codes for the GLYT1 protein, a biochemical abnormality in the GCS, and dihydrolipoamide dehydrogenase enzymes, which function as a GCS component, are responsible for the neonatal form’s symptoms, which include progressive encephalopathy, hypotonia, seizures, and occasionally mortality in the first few days of life. By changing the MAPK signalling pathways, glycine deprivation in the brain damages neurons by increasing NMDA receptor activation, increasing intracellular Ca levels, and leading to DNA breakage and cell death in the neuron region. In addition to the previously mentioned clinical diagnosis, NKH or GE would be determined by MLPA and 13C glycine breath tests. Pediatricians, surgeons, neurologists, and geneticists treat NKH and GE at the newborn period; there is no cure for either condition.