Multiplex Ligation-dependent Probe Amplification Analysis Research Articles

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.

High prevalence of copy number variations in the Japanese participants with suspected MODY.

Maturity-Onset Diabetes of the Young (MODY) is a diabetes mellitus subtype caused by a single gene. The detection rate of the responsible gene is 27% in the United Kingdom, indicating that the causative gene remains unknown in the majority of clinically diagnosed MODY cases. To improve the detection rate, we applied comprehensive genetic testing using whole exome sequencing (WES) followed by Multiplex Ligation-dependent Probe Amplification (MLPA) and functional analyses. Twenty-one unrelated Japanese participants with MODY were enrolled in the study. To detect copy number variations (CNVs), WES was performed first, followed by MLPA analysis for participants who were negative on the basis of WES. Undetermined variants were analyzed according to their functional properties. WES identified 7 pathogenic and 3 novel likely pathogenic variants in the 21 participants. Functional analyses revealed that 1 in 3 variants was pathogenic. MLPA analysis applied to the remaining 13 undetermined samples identified 4 cases with pathogenic CNVs: 3 in HNF4A and 1 in HNF1B. Pathogenic variants were identified in 12 participants (12/21, 57.1%) - relatively high rate reported to date. Notably, one-third of the participants had CNVs in HNF4A or HNF1B, indicating a limitation of WES-only screening.

Cytogenetics investigation in 151 Brazilian infertile male patients and genomic analysis in selected cases: experience of 14 years in a public genetic service

ObjectivesMale infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience.ResultsWe evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: two had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (CDY2B and NLGN4Y genes) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (PRY and RBMY1J genes).

A Pilot Study of Multiplex Ligation-Dependent Probe Amplification Evaluation of Copy Number Variations in Romanian Children with Congenital Heart Defects.

Congenital heart defects (CHDs) have had an increasing prevalence over the last decades, being one of the most common congenital defects. Their etiopathogenesis is multifactorial in origin. About 10-15% of all CHD can be attributed to copy number variations (CNVs), a type of submicroscopic structural genetic alterations. The aim of this study was to evaluate the involvement of CNVs in the development of congenital heart defects. We performed a cohort study investigating the presence of CNVs in the 22q11.2 region and GATA4, TBX5, NKX2-5, BMP4, and CRELD1 genes in patients with syndromic and isolated CHDs. A total of 56 patients were included in the study, half of them (28 subjects) being classified as syndromic. The most common heart defect in our study population was ventricular septal defect (VSD) at 39.28%. There were no statistically significant differences between the two groups in terms of CHD-type distribution, demographical, and clinical features, with the exceptions of birth length, weight, and length at the time of blood sampling, that were significantly lower in the syndromic group. Through multiplex ligation-dependent probe amplification (MLPA) analysis, we found two heterozygous deletions in the 22q11.2 region, both in patients from the syndromic group. No CNVs involving GATA4, NKX2-5, TBX5, BMP4, and CRELD1 genes were identified in our study. We conclude that the MLPA assay may be used as a first genetic test in patients with syndromic CHD and that the 22q11.2 region may be included in the panels used for screening these patients.

Improved sensitivity for detection of pathogenic variants in familial NF2-related schwannomatosis

PurposeTo determine the impact of additional genetic screening techniques on the rate of detection of pathogenic variants leading to familial NF2-related schwannomatosis.MethodsWe conducted genetic screening of a cohort of 168...

Comparison of the accuracy of multiplex digital PCR versus multiplex ligation-dependent probe amplification in quantification of the survival of motor neuron genes copy numbers

For over two decades, multiplex ligation-dependent probe amplification (MLPA) has served as the gold standard for genetic testing of spinal muscular atrophy. However, there is emerging evidence questioning the reliability of MLPA in determining the copy numbers (CNs) of the survival of motor neuron (SMN) gene in certain cases. Recently, digital polymerase chain reaction (dPCR) has shown potential for better performance in copy number variant detection. This study aimed to compare MLPA and dPCR in quantifying SMN1 and SMN2 CNs, identify reasons for observed discrepancies, and explore the clinical implications of false results.A total of 733 DNA samples, previously subjected to MLPA analysis, were tested using multiplex droplet dPCR assays. Samples exhibiting inconsistent results between the two methods underwent repeated dPCR assays. When inconsistencies persisted, a third method was employed for verification. Digital PCR yielded results consistent with those of MLPA in 94.4% (692/733) of samples. Forty-one cases exhibited quantitative disparities in SMN1 and/or SMN2 CNs between the two methods. Confirmatory tests revealed that 37 inaccurate results were produced by the MLPA analysis, whereas four were attributed to the dPCR method. The dPCR technique exhibits better accuracy than MLPA and is qualified for SMA genetic testing across various clinical scenarios.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

Crebbp Mutations Are Associated with Slow Minimal Residual Response to Upfront Induction Chemotherapy in Pediatric High-Hyperdiploid B-Cell Precursor Acute Lymphoblastic Leukemia Treated in the AIEOP-BFM ALL 2009 Protocol

Introduction High-hyperdiploidy (HHD) represents the most common cytogenetic abnormality found in B-cell precursor acute lymphoblastic leukemia (B-ALL). Despite the overall good outcome of this entity, up to 15% of patients suffer from disease relapse. Genome-wide ALL studies with next-generation sequencing (NGS) approaches identified a distinct pattern of somatic mutations in HHD ALL, enriched in mutations occurring in genes activating the RAS pathway and in CREBBP gene, especially in relapsed patients. Although several studies have attempted to explore the prognostic significance of these mutations that are potentially susceptible of targeted therapies, their impact in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus on their clinical significance. In this study, we aimed to investigate the frequency of recurrent mutations and to define their prognostic role in the setting of newly diagnosed HHD B-ALL, by using end of induction minimal residual disease (MRD) and event-free survival (EFS). Methods We included 101 consecutive patients, aged between 1-17 years, with newly diagnosed HHD B-ALL and enrolled in the AIEOP-BFM ALL 2009 protocol (NCT01117441). HHD was defined by the presence of a DNA index ≥1.16 and lack of ETV6::RUNX1, BCR::ABL1, KMT2A::AFF1 translocations. Diagnostic leukemia samples of 89 patients were sequenced using a validated clinical NGS panel profiling 39 genes involved in leukemia pathogenesis. Mutations affecting RAS pathway ( NRAS, KRAS, PTPN11, NF1, BRAF) and mutations affecting CREBBP gene were included. In addition, digital multiplex ligation-dependent probe amplification (digital MLPA) analysis for the identification of IKZF1 deletion and IKZF1plus phenotype was evaluable for 67 patients. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. MRD was assessed by an IG/TR PCR assay at day +33 of induction (timepoint 1, TP1) and at day +78 (timepoint 2, TP2). EFS was estimated by using Kaplan and Meier method and differences between groups were tested using the log-rank test. Resistance to treatment, relapse, death or second malignant neoplasm were considered as events, whichever occurred first. Results Patients' characteristics according to the presence of studied mutations are shown in Table 1. Mutations affecting RAS pathway and CREBBP occurred in 57.3% and 12.3% of HHD B-ALL patients, respectively. Clonal RAS pathway mutations, defined by variant allele frequency (VAF) ≥25%, were found in 16.8% of cases. Among the 11 patients with mutations of CREBBP, 7 had also a mutation affecting KRAS and the association was significant (p=0.03). Seven patients (10.4%) had deletion of IKZF1, of whom 5 (7.4%) were IKZF1plus. There was no association between any mutations or IKZF1 status and patient presenting characteristics, as including sex, age and WBC counts. MRD was evaluable in 84 patients at the end of induction (TP1). MRD level ≥5x10 -4 was found in 36.4% of patients with CREBBP mutations and in 9.6% of those without, and this association was statistically significant (p=0.02). Additionally, MRD negativity was found in 9.1% and 38.4%, respectively. At TP2, MRD was still detectable in 45.5% of patients with CREBBP mutations compared to 20.6% of those with CREBBP wildtype (p=0.08). Similarly, we observed an association between RAS pathway mutations and MRD at TP1 (p=0.01) with a marked difference in MRD negative patients (21.3% vs 51.4% in patients with and without mutations). No association was observed between MRD level and clonal RAS pathway mutations or IKZF1 status. Despite the association between CREBBP mutations and high MRD at TP1, no significant difference in final high-risk group was found (27.3% vs 11.5%, p=0.17). A total of 8 events were observed, of which 6 were relapses. No impact of any mutations or IKZF1 status in term of 5-years EFS was found in our cohort. Conclusion Our study shows that CREBBP mutations in HHD B-ALL are associated with inferior early response to treatment in terms of MRD level at the end of induction (TP1) but not with worse EFS with our whole current chemotherapy backbone. Therefore, the inclusion of CREBBP mutated patients in very low risk protocols should be carefully evaluated. The validation of these findings is ongoing through the analysis of patients treated by other international study groups adopting different chemotherapy backbones.

Adverse Prognostic Role of Copy Number Alterations and Mutations in Adults with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia

Background: The impact of copy number alterations (CNAs) or mutations on outcomes has been broadly analyzed in pediatric acute lymphoblastic leukemia (ALL). However, the magnitude of the effect was often marginal, frequently restricted to specific genetic subtypes and rarely replicated across independent cohorts in adult ALL. Aim: We tried to analyze the prognostic relevance of genetic copy number alterations and gene mutations in patients with Ph-negative ALL who were treated with modified hyper-CVAD based chemotherapy followed by allogeneic hematopoietic cell transplantation (alllo-HCT). Methods: We investigated the role of CNAs or mutations to refine risk stratification in 128 adults with newly diagnosed Philadelphia chromosome (Ph)-negative B-cell precursor ALL treated with intensive chemotherapy followed by allo-HCT for post-remission therapy. We performed multiplex ligation-dependent probe amplification (MLPA) to detect deletions of 11 genes ( IKZF1, CDKN2A/B, EBF1, ETV6, PAX5, BTG1, JAK2, RB1, PAR1, ZFY) and high throughput sequencing (HTS) of 70 gene mutations. Results: MLPA analysis showed that IKZF1 and CDKN2A/B deletions were observed in 54 (42.2%) and 52 (40.6%) patients, respectively. The remaining gene deletions were: PAX5 in 29 (22.6%), ETV6 in 22 (17.2%), BTG1 in 15 (11.7%), EBF1 in 14 (10.9%), RB1 in 13 (10.1%), JAK2 in 8 (6.2%), and PAR1 in 4 (3.1%). By HTS, NRAS (n=21, 16.4%), KRAS (n=11, 8.6%), TP53 (n=14, 10.9%), and PTPN11 (n=12, 9.4%) mutations were frequently detected. Overall, multivariate analysis for disease-free survival (DFS) and cumulative incidence of relapse (Table 1) showed that TP53 mutations (HR 5.04, 95%CI 2.29-11.30, P<0.001) and CDKN2A/B deletions (HR 1.88, 95%CI 1.10-3.31, P=0.026) were significantly associated with a poorer DFS. Patients with TP53 mutations (HR 5.64, 95%CI 1.95-16.20, P<0.001) and IKZF1 deletions (HR 1.91, 95%CI 1.09-3.31, P=0.022) had a higher relapse risk. To better define the prognostic role of CNAs, further analyses were restricted to patients excluding 14 TP53 mutations (n=114). Here, we classified patients into 4 subgroups; (1) No deletions (n=45, 39.5%), (2) IKZF1 deletion-alone (n=11, 9.6%), (3) IKZF1plus (n=40, 35.1%), and (4) CDKN2A/B±other deletions without IKZF1 deletions (n=18, 15.8%). Patients with IKZF1plus or CDKN2A/B±other deletions had an inferior DFS ( P=0.023) and higher relapse incidence ( P=0.002) than patients having no deletions. The results were similarly reproduced in patients receiving allo-HCT in first complete remission. Conclusions: Our data showed that IKZF1 or CDKN2A/B deletions and TP53 mutations may confer a poor prognosis for adult Ph-negative B-cell precursor ALL in the setting of intensive chemotherapy and allo-HCT. The combination of genomic abnormalities and minimal residual disease response may further refine risk stratification and better select patients who could benefit from novel therapeutic approaches.

Target-Oriented Classification of Triple-negative Breast Cancer.

Breast cancer that is estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and human epidermal growth factor receptor-2 (HER2)-negative is termed triple-negative breast cancer (TNBC). Cytotoxic chemotherapy remains the first choice of treatment against TNBC due to lack of specific therapeutic targets. TNBC is not classified based on therapeutic targets, but recently, the development of targeted therapies - including immune checkpoint inhibitors and poly (adenosine diphosphate-ribose) polymerase inhibitors - has gained attention. This study aimed to examine a novel target-oriented TNBC classification to further facilitate targeted therapy by classifying TNBC based on the breast cancer 1 (BRCA1)-like as well as the protein expression of HER2, programmed death ligand 1 (PD-L1), androgen receptor (AR), cytokeratin 5/6, and epidermal growth factor receptor (EGFR). We enrolled 17 patients with primary TNBC who did not receive preoperative chemotherapy and underwent surgery at the Kobe University Hospital, Japan, between January 1, 2018, and July 31, 2019. Immunohistochemical staining was performed on tumor specimens, while a BRCAness test was performed using multiplex ligation-dependent probe amplification (MLPA) analysis. A BRCAness score 0.5 or higher was considered BRCA1-like. Tumors were classified as HER2-low (immunohistochemistry score 1+ or 2+ and FISH negative), PD-L1 positive, AR positive, or BRCA1-like. HER2-low, PD-L1 positive, AR positive, and BRCA1-like were detected in 11 (64.7%), 4 (23.5%), 6 (35.3%), and 6 (35.3%) samples. The tumor of only one patient could not be classified into any of these categories. Almost all TNBC cases can be classified according to treatable targets.

Uniparental disomy: expanding the clinical and molecular phenotypes of whole chromosomes.

Uniparental disomy (UPD) refers to as both homologous chromosomes inherited from only one parent without identical copies from the other parent. Studies on clinical phenotypes in UPDs are usually focused on the documented UPD 6, 7, 11, 14, 15, and 20, which directly lead to imprinting disorders. This study describes clinical phenotypes and genetic findings of three patients with UPD 2, 9, and 14, respectively. Chromosomal microarray (CMA), UPDtool, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and whole-exome sequencing (WES) analysis were performed to characterize the genetic etiology. The CMA revealed a homozygous region involving the whole chromosome 2 and 9, a partial region of homozygosity in chromosome 14. UPD-tool revealed a paternal origin of the UPD2. MS-MLPA showed hypomethylation of imprinting gene MEG3 from maternal origin in the UPD14 case. In addition, UPD14 case displayed complex symptoms including growth failure, hypotonia and acute respiratory distress syndrome (ARDS), accompanied by several gene mutations with heterozygous genotype by WES analysis. Furthermore, we reviewed the documented UPDs and summarized the clinical characteristics and prognosis. This study highlighted the importance to confirm the diagnosis and origin of UPD using genetic testing. Therefore, it is suggested that expanding of the detailed phenotypes and genotypes provide effective guidance for molecule testing and genetic counseling, and promote further biological investigation to the underlying mechanisms of imprinted disorders and accompanied copy number variations.

SDPS-27 P16 IMMUNOHISTOCHEMISTRY AS A SCREENING TOOL FOR HOMOZYGOUS CDKN2A DELETIONS IN GLIAL TUMORS

Abstract The 2021 WHO classification of tumors of the central nervous system emphasizes the significance of molecular parameters for an integrated diagnosis. Homozygous deletion of CDKN2A has been associated with an adverse prognosis in IDH-mutant gliomas, supratentorial ependymomas, meningiomas, and MPNST. In this study, we examined the value of p16 protein immunohistochemistry as a screening tool for predicting a homozygous CDKN2A deletion. Genetic analyses for CDKN2A deletion in 30 pleomorphic xanthoastrocytomas, 32 IDH-wildtype high-grade gliomas and 40 supratentorial ependymomas with ZFTA-RELA gene fusion, 10 IDH-mutant astrocytomas, and 10 meningiomas were performed either by a molecular inversion probe assay (MIP), a high-resolution, quantitative technology for the accurate, genome-wide assessment of chromosomal copy number alterations, or by 850k methylation profile-deduced copy number analysis or multiplex ligation-dependent probe amplification analysis. In pleomorphic xanthoastrocytomas and IDH-wildtype high-grade gliomas, p16 immunohistochemistry showed high specificity (100% both), moderate sensitivity (76% and 56%), and a high positive predictive value (PPV; 100% both) for the presence of a homozygous CDKN2A deletion. In supratentorial ependymoma with ZFTA-RELA gene fusion, immunohistochemistry for p16 protein was highly sensitive and specific (90% and 97%) for a homozygous CDKN2A deletion and exhibited a high PPV and NPV (90% and 97%, respectively). For lower-grade meningioma, no correlation between the p16 immunohistochemical status and the CDKN2A deletion status was possible. Therefore, p16 protein expression detected by immunohistochemistry is a useful prescreening tool for detecting a homozygous CDKN2A deletion in glial tumors.

Copy Number Variations in Hereditary Spastic Paraplegia-Related Genes: Evaluation of an Iranian Hereditary Spastic Paraplegia Cohort and Literature Review

Introduction: In human genetic disorders, copy number variations (CNVs) are considered a considerable underlying cause. CNVs are generally detected by array-based methods but can also be discovered by read-depth analysis of whole-exome sequencing (WES) data. We performed WES-based CNV identification in a cohort of 35 Iranian families with hereditary spastic paraplegia (HSP) patients. Methods: Thirty-five patients whose routine single-nucleotide variants (SNVs) and insertion/deletion analyses from exome data were unrevealing underwent a pipeline of CNV analysis using the read-depth detection method. Subsequently, a comprehensive search about the existence of CNVs in all 84 known HSP-causing genes was carried out in all reported HSP cases, so far. Results and Discussion: CNV analysis of exome data indicated that 1 patient harbored a heterozygous deletion in exon 17 of the SPAST gene. Multiplex ligation-dependent probe amplification analysis confirmed this deletion in the proband and his affected father. Literature review demonstrated that, to date, pathogenic CNVs have been identified in 30 out of 84 HSP-causing genes (∼36%). However, CNVs in only 17 of these genes were specifically associated with the HSP phenotype. Among them, CNVs were more common in L1CAM, PLP1, SPAST, SPG7, SPG11, and REEP1 genes. The identification of the CNV in 1 of our patients suggests that WES allows the detection of both SNVs and CNVs from a single method without additional costs and execution time. However, because of intrinsic issues of WES in the detection of large rearrangements, it may not yet be exploited to replace the CNV detection methods in standard clinical practice.

Prenatal detection of copy number variants in fetuses with detected congenital devolpmental disordes, from 2015 to 2020 by Multiplex Ligation-Dependent Probe Amplification and microarray analysis.

Analysis of prenatal samples from 2015 to 2020. Comparison detection rates of clinically relevant variants by cytogenetic karyotype analysis and cytogenomic MLPA (Multiplex Ligation-Depent Probe Amplification) and microarray methods (CMA - chromosomal microarray). 1,029 prenatal samples were analyzed by cytogenetic karyotyping (N = 1,029), cytogenomic methods - MLPA (N = 144) and CMA (N = 111). All unbalanced changes were confirmed by MLPA or CMA. From the analyzed set of fetuses, after subtraction of aneuploidies - 107 (10.40%, N = 1,029), 22 structural aberrations (2.39%, N = 922) - nine unbalanced changes (0.98%), 10 balanced changes (1.08%), one case of unclear mosaicism (0.09%), one case of presence of a marker chromosome (0.09%) and one case of sex discordance (0.09%) - were detected by karyotype analysis. A total of eight (7.21%, N = 111) pathological variants were detected by CMA in 255 samples with physiological karyotype indicated for cytogenomic examination. Five (3.47%, N = 144) of eight pathogenic variants were detected by MLPA method. The total capture of pathogenic variants by MLPA and CMA methods was 14 (5.14%) and 17 (6.25%) (N = 272), including confirmatory pathological karyotype testing. Detection of pathological variants in the isolated disorders group was lower than in the multiple disorders group (5.08 vs. 21.42%). A higher success rate for the detection of pathological copy number variation variants by the microarray method than by the MLPA method was confirmed.

#4485 CLINICAL CASE OF MOSAICISM IN ADPKD PATIENT

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) it's mainly characterized by renal involvement with progressive bilateral development of renal cysts and volumetric increase of the kidneys, responsible for the progressive loss of renal function that leads to chronic kidney disease (CKD) and the need of renal replacement therapy. There are some cases (doubtful, uncertain) in which it is necessary to perform genetic testing to achieve a definitive diagnosis. It looks like in ADPKD patients, mosaicism could modulate the clinical course of the disease. Mosaicism is characterized by few cell populations with different genomes. In these special cases the diagnosis is very difficult and the genetic analysis could be an important and helpful tool. With this report, we want to highlight the importance of the use of several diagnostic methods in those ‘atypical’ cases where otherwise the analysis would have been negative from a first molecular approach. Method A 47 years old woman came to our attention with typical picture of ADPKD, arterial hypertension and chronic kidney disease CKD (3b). She had no ADPKD family history, but since she had a son, genetic testing was performed. According to our workflow, first level analysis includes Next Generation Sequencing (NGS). Second level analysis, performed to investigate other genes and large rearrangement, was based on Multiplex Ligation-dependent Probe Amplification (MLPA) analysis of PKD1 and PKD2 genes, using Salsa MLPA ProbeMix P352 PKD1PKD2 kit and ProbeMix P351 PKD1 kit ©MRC Holland. Results NGS did not detect the presence of any genomic variants. Therefore, second-level genetic analysis (several approaches), showed a large deletion in heterozygosis of the portion comprising the 3-33 exons of PKD1 gene in mosaic in percentage close to the resolution limits of the used technique (25-30%). The same analysis was confirmed in another laboratory. Conclusion We concluded that the identified large deletion, without establishing the real borders due to MLPA technical restrictions, was present as mosaicism. The variant is not reported, but due to the type of mutation and the clinical picture of the patient, it is to be considered as likely pathogenic. A stepwise genetic approach might be useful in those cases where standard methods do not allow to reach a definitive diagnosis.

Terminal microdeletion of chromosome 18 in a Malaysian boy characterized with few features of typical 18q- deletion syndrome: a case report

BackgroundThe 18q- deletion syndrome is a rare congenital chromosomal disorder caused by a partial deletion of the long arm of chromosome 18. The diagnosis of a patient with this syndrome relies on the family medical history, physical examination, developmental assessment, and cytogenetic findings. However, the phenotype of patients with 18q- deletion syndrome can be highly variable, ranging from almost normal to severe malformations and intellectual disability, and normal cytogenetic findings are common, thus complicating the diagnosis. Interestingly, only few characteristic features of typical 18q- deletion syndrome were found in the patient, despite sharing the same critical region. To our knowledge, this is the first report of a Malaysian individual with 18q- terminal microdeletion diagnosed with microarray-based technology.Case presentationHere we report a 16-year-old Malaysian Chinese boy, a product of a non-consanguineous marriage, who presented with intellectual disability, facial dysmorphism, high arched palate, congenital talipes equinovarus (clubfoot), congenital scoliosis, congenital heart defect, and behavioral problems. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XY G-banded karyotype. Array-based comparative genomic hybridization was performed using a commercially available 244 K 60-mer oligonucleotide microarray slide according to the manufacturer’s protocol. This platform allows genome-wide survey and molecular profiling of genomic aberrations with an average resolution of about 10 kB. In addition, multiplex ligation-dependent probe amplification analysis was carried out using SALSA MLPA kit P320 Telomere-13 to confirm the array-based comparative genomic hybridization finding. Array-based comparative genomic hybridization analysis revealed a 7.3 MB terminal deletion involving chromosome band 18q22.3-qter. This finding was confirmed by multiplex ligation-dependent probe amplification, where a deletion of ten probes mapping to the 18q22.3-q23 region was detected, and further multiplex ligation-dependent probe amplification analysis on his parents showed the deletion to be de novo.ConclusionThe findings from this study expand the phenotypic spectrum of the 18q- deletion syndrome by presenting a variation of typical 18q- deletion syndrome features to the literature. In addition, this case report demonstrated the ability of the molecular karyotyping method, such as array-based comparative genomic hybridization, to assist in the diagnosis of cases with a highly variable phenotype and variable aberrations, such as 18q- deletion syndrome.

The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes.

The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype). 38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay. Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH). The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.

Correlation between genotype and phenotype with special attention to hearing in 14 Japanese cases of NF2-related schwannomatosis

NF2-related schwannomatosis (NF2) is an autosomal dominant genetic disorder caused by variants in the NF2 gene. Approximately 50% of NF2 patients inherit pathogenic variants, and the remainder acquire de novo variants. NF2 is characterized by development of bilateral vestibular schwannomas. The genetic background of Japanese NF2 cases has not been fully investigated, and the present report performed a genetic analysis of 14 Japanese NF2 cases and examined genotype–phenotype correlations. DNA samples collected from peripheral blood were analyzed by next-generation sequencing, multiplex ligation-dependent probe amplification analysis, and in vitro electrophoresis. Ten cases had pathogenic or likely pathogenic variants in the NF2 gene, with seven truncating variants and three non-truncating variants. The age of onset in all seven cases with truncating variants was < 20 years. The age of onset significantly differed among cases with truncating NF2 variants, non-truncating NF2 variants, and no NF2 variants. However, the clinical course of tumor growth and hearing deterioration were not predicted only by germline pathogenic NF2 variants. The rate of truncating variants was higher in the present study than that of previous reports. Genotype–phenotype correlations in the age of onset were present in the analyzed Japanese NF2 cases.

A novel deletion of exon 4 in the Ectodysplasin A gene associated with X-linked hypohidrotic ectodermal dysplasia

ObjectiveIdentify the disease-causing mutation in a patient with features of X-linked hypohidrotic ectodermal dysplasia, which is a genetic disorder characterized by hypodontia, hypohidrosis and hypotrichosis. It is caused by mutations in Ectodysplasin A gene, which encodes ectodysplasin A, a member of the tumor necrosis factor superfamily. DesignGenetic analysis, was performed using chromosomal microarray analysis, whole exome sequencing and multiplex ligation-dependent probe amplification analysis in a 4-year-old boy with hypohidrotic ectodermal dysplasia features. Moreover, the boy’s parents were tested for clinically significant findings identified in order to elucidate the pattern of inheritance of the finding detected in the proband. ResultsA novel deletion of entire exon 4 in Ectodysplasin A gene identified in the 4-year-old patient. This deletion was found in heterozygous state in the mother of the proband and was not detected in his father. RNA analysis revealed an in-frame deletion r.527_706del, p.(176_236del) in exon 4 of the Ectodysplasin A gene. ConclusionWe identified a novel gross deletion in the Ectodysplasin A gene in a male patient with X-linked hypohidrotic ectodermal dysplasia. Clinical and molecular genetic analysis are crucial to set an accurate diagnosis in patients with hypohidrotic ectodermal dysplasia. These results highlight the importance of the collagen domain of Ectodysplasin A, encoded by exon 4, for its function in vivo.

Mutation spectrum and frequency of copy number variations of the ANOS1 gene in patients with Kallmann syndrome or normosmic isolated hypogonadotropic hypogonadism.

This study was performed to investigate the molecular characteristics and frequency of copy number variations (CNVs) of ANOS1 in patients with Kallmann syndrome (KS) or normosmic isolated hypogonadotropic hypogonadism (nIHH) using multiplex ligation-dependent probe amplification (MLPA) analysis and sequencing. Among 45 patients from 43 independent families, Sanger sequencing, next-generation sequencing (NGS), or microarray was performed in 24 patients from 23 families, and MLPA was performed in 19 patients who did not show rare sequence variants (n = 18) or ANOS1 amplification by PCR (n = 1). Seven patients (4 patients with KS, 1 patient with nIHH, one prepubertal boy with anosmia, and one newborn patient) from 6 families (6/43, 14%) harbored molecular defects in ANOS1 including a nonsense mutation [c.1140G>A (p.W380*)], a frameshift mutation [c.1260del (p.Q421Kfs*61)], a splice site mutation (c.1449+1G>A), an exon 7 deletion, a complete deletion, and 7.9 Mb-sized inversion encompassing ANOS1. The complete deletion of ANOS1 was identified in a neonate with a micropenis and cryptorchidism. Unilateral renal agenesis was found in three patients, whereas only one patient displayed both synkinesia and sensorineural hearing loss. There was no reversal of hypogonadotropic hypogonadism in any patient during 9.1 ± 2.9 years of treatment with testosterone enanthate. Molecular defects in the ANOS1 gene could be identified in 14% of probands including various types of CNVs (3/43, 7.0%). Comprehensive analysis using sequencing and analysis for CNVs is required to detect molecular defects in ANOS1.