Multiplex Ligation-dependent Probe Amplification Research Articles

Contribution of clinically relevant copy number variations in long QT syndrome: a large cohort study

Abstract Background Copy number variations (CNVs) have increasingly been recognized in the cardiovascular setting. Their role in hypertrophic cardiomyopathy or sudden cardiac death has been described, but their involvement in Long QT syndrome (LQTS) has been scarcely studied. Most of these descriptions are confined to case reports or small series. Purpose The aim of this study is to investigate the proportion of clinically relevant CNVs among a large cohort of index cases referred for genetic testing due to LQTS. Methods Retrospective study in which the presence of likely pathogenic (LP) or pathogenic (P) CNVs was evaluated in a cohort of index cases referred for genetic study with LQTS phenotype. Index cases were genotyped with customized libraries through next generation sequencing (NGS). CNVs analysis was conducted by a proprietary method by means of comparative analysis of normalized read depth data. Clinically relevant (LP or P) CNVs were then confirmed by other techniques. Results 1183 index cases were referred for genetic testing to our laboratory with long QT syndrome phenotype. Some of these index cases were referred for suspicion of Jervell-Lange-Nielsen syndrome (JNLS) or for LQTS associated with congenital deafness (n=10) or Anderson-Tawil syndrome (n=1). Around 27% (n=322) of the index cases had a positive result. CNVs analysis was run on 1071 (91%) of the index cases. Most of the CNVs were considered negative (n=994, 93%), 59 could not be analysed (6%) and in 11 patients (1%), a LP/P CNV was identified. All CNVs but three (72.72%), were confirmed by orthogonal molecular techniques (Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA)). In all, but two, the genetic study was considered positive due to the CNV findings. One of these patients carried a pathogenic lost-of-function (LOF) CNV in the LDLR gene (incidental finding) but the report was negative because he was referred for suspicion of JLNS. In another case a CNV in the DSP gene was identified in mosaicism (30-40%). However, in this case, the positive report was attributed to the presence of an additional pathogenic variant in CACNA1C. Regarding the other index cases with LP/P CNVs: 4 carried a deletion in the KCNH2 gene, 3 carried a deletion in KCNQ1 gene (one of them in homozygosis and was studied for suspicion of JLNS) and 1 carried a duplication in the KCQN1 (LOF variant). In addition, one patient carried a deletion of exon 3 in the RYR2 gene. Even if the patient was referred for suspicion of LQTS, the report was considered positive. These clinically relevant CNVs represented around 3% of the positive genetic results. Conclusions In our large cohort of patients referred for genetic testing for LQTS, around 1% of clinically relevant CNVs were identified. As such large genomic rearrangements underlie a non-neglectable portion of cases, we consider that their analysis should be performed as part of the routine genetic testing.Deletion exons 6-14 in KCNH2 gene (CNV)MLPA with deletion exons 6-14 in KCNH2

#1558 Mutational spectrum of X-linked hypophosphatemia (XLH) in Hungarian patients

Abstract Background and Aims X-linked hypophosphatemia (XLH), the most common form of hypophosphatemic rickets, is caused by genetic alterations in the PHEX gene. Prevalence of the X-linked disease is ∼1/20,000. The PHEX gene has 22 exons and encodes a 749 amino acid protein with homology to zinc metallopeptidases. The physiological protein target for PHEX cleavage is unknown. PHEX promotes mineralization by regulating the expression of FGF23. To date, more than 800 disease-causing variants in the PHEX gene have been described in XLH patients. These variants might occur across the entire gene and include missense, nonsense, frameshift, splice site, copy-number variants with a consequence of loss of PHEX protein function. Genetic confirmation of XLH has significant clinical importance from risk prediction to modern treatment options. Here we describe the genetic results of 20 mutation-positive patients from 18 unrelated families, the first such report from Hungary. Method The cohort consisted of 8 male and 12 female patients (mean age at genetic diagnosis (range): 8.25 years (3-17 years) and 12 years (2-32 years)). Genomic DNA was isolated from peripheral blood leukocytes and in the mosaic case also from mouthwash sample. PHEX sequencing was performed using next generation sequencing with a custom designed library preparation kit (Twist Biosciences) on an Illumina MiSeq instrument. For the bioinformatic analysis, NextGene software was used. All small-scale mutations were confirmed by Sanger sequencing. For copy number variation (CNV) analysis, a coverage-based pipeline was used. Detected CNVs were confirmed using multiplex ligation-dependent probe amplification (MLPA). Mutation classification was performed according to the ACMG guidelines. Results All female patients were heterozygous, while all but one male patients were hemizygous. In case of one male patient, a mosaic deletion of exon 10 was observed. Most common mutation type was nonsense (n = 7) followed by small deletions/insertions (n = 6) and splicing alterations (n = 3). One missense mutation was detected. CNVs were responsible in three cases (two large deletions and one large duplication). From the 11 most common PHEX alleles known from the literature (Hum Mutat 2022;43:143-57), three (c.2104C > T, c.2239C > T, c.871C > T) were detected in our cohort. Nine novel, previously undescribed alterations were found: c.264G > A, c.1073_1074delGA, c.1148dupA, c.1645+1G > C, c.1682G > A, c.1701-1G > C, c.2150T > G, c.2172delT, e6-15dup. Conclusion Our data shed more light on the distribution of the PHEX causative variations in the Hungarian XLH patients and opens the possibility of the state-of-the-art treatment in many of them.

#1232 Integrating genotype and phenotype data is required for renal survival prediction in autosomal dominant polycystic kidney disease

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the cause for 5-10% of end stage kidney disease (ESKD) worldwide. Accurate renal prognosis is essential for effective disease management. Investigation of the prognostic value of combining genetic and phenotypic characteristics in a cohort of ADPKD patients is attempted. Method In 66 ADPKD patients (mean age 33 ± 12 years, 32 males) the genetic diagnosis (using targeted Next Generation, tNGS, or Sanger Sequencing or Multiplex ligation-dependent probe amplification, MLPA) and the Mayo Clinic Imaging Category (MCIC) (through Renal Magnetic Resonance Imaging, MRI, for Total Kidney Volume measurement) were assessed. The prediction of ESKD was performed using the Mayo Clinic formula (Irazabal MV et al JASN, 2015). The calculation incorporated variables such as sex, age, e-GFR, and the MCIC. The PROPKD score was derived by considering both phenotypic factors (sex, hypertension and urologic events before the age of 35 years) and genetic data (PKD1-truncating, PKD1-nontruncating and PKD2). Results Adjusted for age, the e-GFR was on average, 26 and 27 ml/min higher in PKD2 patients compared to ADPKD-PKD1-truncating and ADPKD-PKD1-non-truncating patients, respectively (p = 0.001). The median prediction of ESKD for ADPKD-PKD1-truncating, ADPKD-PKD1-non-truncating and PKD2 patients was 22 (11-31) years, 22 (11-34) years and 31 (21-59) years, respectively (NS). In patients categorized as high risk according to PROPKD (score 7-9) the predicted time to ESKD was 16 (9-30) years, for those at intermediate risk (score 4-6), 25 (13-34) years and for those at low risk (score 0-3), 28 (16-55) years (NS). Finally, the PROPKD score showed an inverse correlation to the years of ESKD prediction, as determined by the Mayo Clinic formula; a higher PROPKD score was associated with a shorter prediction period for ESKD (Spearman r = −0.3, p = 0.04). Conclusion Prognosis of renal survival in ADPKD patients requires not only a specific genetic diagnosis but also consideration of the phenotypic data, as incorporated in the PROPKD score, enhancing the accuracy of renal survival prediction.

#191 An unusual cause of hypocalcemia in a young patient

Abstract Background and Aims A 33-year-old man visited the Nephrology Department outpatient clinic with abnormal laboratory findings: his serum calcium level was 5.9 mg/dL in a recent health examination. He denied any underlying disease and was on no medications. He complained of a tingling sensation in his hands and of eyelid twitching. On physical examination, he showed Chvostek's sign. His laboratory findings including serum calcium level are shown in Table 1. He did not have any morphological abnormalities, such as a round face, brachydactyly, or dental anomalies. Method Since he showed hypocalcemia, normal 25-hydroxyvitamin D, and elevated parathyroid hormone (PTH) levels, the presence of PTH resistance was presumed. Under strong suspicion of pseudohypoparathyroidism (PHP) in this patient, DNA sequence analysis and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of GNAS and STX16, genetic regions that are often affected by mutations or imprinting changes in PHP, was performed. No pathogenic mutations in GNAS and STX16 were detected by sequencing. Results Results of MS-MLPA of the GNAS locus demonstrated abnormal methylation patterns in the patient after HhaI restriction enzyme treatment. The methylation ratio showed a gain of methylation in the NESP55 region and loss of methylation in the AS, XL, and A/B differentially methylated regions (DMRs) in the patient as compared to a healthy control (Fig. 1). The patient was diagnosed with PHP type 1B (PHP1B) due to the methylation abnormalities in multiple DMRs. His parents both showed normal serum calcium levels; thus, no further genetic testing was conducted. He is on outpatient follow-up with adequate calcium replacement and no specific symptoms. Conclusion PHP is a rare disorder of unknown prevalence. It is characterized by hypocalcemia, hyperphosphatemia, and increased PTH levels. PHP1B is a subtype of PHP characterized by isolated renal PTH resistance, usually without physical abnormalities such as Albright hereditary osteodystrophy or other endocrine abnormalities. PHP1B can be sporadic or familial. The genetic characteristics of most sporadic PHP1B remains unclear, but GNAS imprinting abnormalities and hypomethylation at the A/B DMRs have been reported. Symptoms of PHP1B usually begin in childhood due to low calcium levels and may include numbness, seizures, tetany, cataracts, and dental problems. However, as in our case, specific symptoms may not occur until adulthood. Therefore, when evaluating the cause of hypocalcemia, it is necessary to differentiate PHP1B, even in adults, and particularly in cases with hyperphosphatemia and high PTH levels.

#2412 Monoallelic NEK8-related polycystic kidney disease in an Irish ADPKD cohort

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), commonly caused by pathogenic variants in PKD1 and PKD2 genes, leads to enlarged cystic kidneys and typically present in adulthood where ∼70% of affected patients progress to kidney failure by their 7th decade of life. Recently, new genes have been reported that appear to explain some of the previously unresolved ADPKD-like cases. An example is the NEK8 gene, where monoallelic variants are reported to cause early-onset ADPKD with progressive phenotype. We are reporting 4 Irish families with monoallelic NEK8 variants. Method In-depth clinical and radiological evaluations were conducted on 250 families with polycystic kidney disease referred to the Irish Kidney Gene Project. All patients underwent genomic testing using gene-panel and exome Next Generation Sequencing (NGS), while multiple ligation probe amplification analysis of PKD1 or PKD2 was utilized in genetically unresolved families. The American College of Medical Genetics and Genomics guidelines were applied to assess variant pathogenicity. Results To date, 471 individuals representing 250 distinct families (F) have been studied, including 105 (42%) families with two or more individuals (326 individuals). 55.2% have reached end-stage kidney failure, with an average age of 49.1 ± 14.3 years. We identified pathogenic / likely pathogenic variants in 76% (190/250) of families, with variants in PKD1 accounting for 76.8% (146/190) and PKD2 accounting for 15.3% (29/190) of families, respectively. In familial cases with non-PKD1/PKD2 variants, we identified pathogenic variants in a small proportion of families with ADPKD-like phenotypes: IFT140 variants (n = 4), ALG5 (n = 2), 17q12 microdeletion (n = 2) and DNAJB11, ALG8, and ALG9 variants (n = 1 each). Upon thorough review of all genetically unresolved PKD families, 4 (6.7%) families were identified with monoallelic protein-kinase domain NEK8 variants [NM_178170.3]: p.R45W (in 2 families) and two other missense variants: p.N69D and p.R140L, none of which contained a second NEK8 variant despite extensive analysis. Autosomal dominant segregation was confirmed in two parent-child pairs, whereas the remaining two index patients reported no family history of kidney disease. The median age at the initial presentation was 12 years (IQR: 2.5–27.3), with a median follow-up period of 22.5 years at the last evaluation. The phenotype of all patients was consistent with polycystic kidney disease with the absence of liver and pancreatic cysts. All patients were diagnosed with hypertension at an average age of 15.3 ± 12.2 years. Cerebral aneurysms were detected in two patients (F1: at age 61 years and F4: at age 54 years), while F3 was reported to have childhood-onset frontal bossing, prominence cerebrospinal fluid, and large arachnoid cysts. Approximately 67% [4/6] patients progressed to end-stage kidney failure by the 3rd decade of life. Compared to families with PKD1/PKD2 variants, families with NEK8 variants were characterized by earlier average age at initial presentation (14.5 versus 29.9 years; p 0.01) and progression to end-stage kidney failure (15.5 versus 49.6 years; p < 0.0001). Table 1 summarizes the clinical and genetic characteristics of the reported families. Conclusion We provide further evidence that monoallelic NEK8 variants cause early presentation and progressive ADPKD-like phenotype. Sequencing for monoallelic NEK8 variants in patients with childhood and early-adulthood cystic kidney disease can inform renal prognosis, treatment planning, familial screening and reproductive choices.

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.

Large regions of homozygosity in prenatal diagnosis.

Chromosomal microarrays (CMA) incorporate single nucleotide polymorphisms to enable the detection of regions of homozygosity (ROH). Here, we retrospectively analyzed 6288 prenatal cases who performed CMA to explored the clinical implications of large ROH in prenatal diagnosis. We analyzed cases with ROH larger than 10 megabases and reviewed the ultrasound findings; karyotype results and pregnancy follow-up data. Cases with possible imprinting disorders were assessed by methylation-specific multiplex ligation-dependent probe amplification. In total, we identified 50 cases with large ROH and chromosomes 1 and 2 were the most affected. About 59.18% of the ROH cases had ultrasound abnormalities, with the most common findings being ultrasound soft-marker abnormalities. There were seven fetuses had ROH which covered almost the entire chromosome and four had terminal ROH that involved almost the entire long arm of the chromosomes, which indicated uniparental disomy (UPD), of which 70% showed abnormal ultrasound findings. Ten cases with multiple ROH on different chromosomes indicated the third to fifth degree of consanguinity. In this study, we highlighted the clinical relevance of large ROH related to UPD. The analysis of ROH allowed us to gain further understanding of complex cytogenetic and disease mechanisms in prenatal diagnosis.

Application of whole exome sequencing in the diagnosis of muscular disorders: a study of Taiwanese pediatric patients

BackgroundMuscular dystrophies and congenital myopathies encompass various inherited muscular disorders that present diagnostic challenges due to clinical complexity and genetic heterogeneity.MethodsThis study aimed to investigate the use of whole exome sequencing (WES) in diagnosing muscular disorders in pediatric patients in Taiwan. Out of 161 pediatric patients suspected to have genetic/inherited myopathies, 115 received a molecular diagnosis through conventional tests, single gene testing, and gene panels. The remaining 46 patients were divided into three groups: Group 1 (multiplex ligation-dependent probe amplification–negative Duchenne muscular dystrophy) with three patients (6.5%), Group 2 (various forms of muscular dystrophies) with 21 patients (45.7%), and Group 3 (congenital myopathies) with 22 patients (47.8%).ResultsWES analysis of these groups found pathogenic variants in 100.0% (3/3), 57.1% (12/21), and 68.2% (15/22) of patients in Groups 1 to 3, respectively. WES had a diagnostic yield of 65.2% (30 patients out of 46), detecting 30 pathogenic or potentially pathogenic variants across 28 genes.ConclusionWES enables the diagnosis of rare diseases with symptoms and characteristics similar to congenital myopathies and muscular dystrophies, such as muscle weakness. Consequently, this approach facilitates targeted therapy implementation and appropriate genetic counseling.

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.

Identification of rare and novel PHEX variants in X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a rare metabolic bone disease caused by inactivation mutations in the PHEX gene. Despite the extensive number of reported PHEX variants, only a few cases of chromosomal abnormalities have been documented. We aimed to identify the pathogenic variants in six unrelated families with a clinical diagnosis of XLH and to propose a genetic workflow for hypophosphatemia patients suspected of XLH. Multiple genetic testing assays were used to analyze the six families' genetic profiles, including whole exome sequencing, multiplex ligation-dependent probe amplification, whole genome sequencing, reverse transcript polymerase chain reaction, Sanger sequencing, and karyotyping. The study identified six novel pathogenic variants, including one mosaic variant (exon 16-22 deletion), three chromosomal abnormalities (46, XN, inv[X][pter→p22.11::q21.31→p22.11::q21.31 →qter], 46, XN, inv[X][p22.11p22.11], and XXY), a nonclassical intron variant (NM_000444.6, c.1701_31A > G), and a deletion variant (NM_000444.6, c.64_5464-186 del5215) of PHEX. Additionally, a genetic testing workflow was proposed to aid in diagnosing patients suspected of XLH. Our research expands the mutation spectrum of PHEX and highlights the significance of utilizing multiple genetic testing methods to diagnose XLH.

Hemizygous deletion of CDKN2A/B with p16 immuno-negative and methylthioadenosine phosphorylase retention predicts poor prognosis in IDH-mutant adult glioma

Abstract Background Homozygous deletion of the tumor suppression genes cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) is a strong adverse prognostic factor in IDH-mutant gliomas, particularly astrocytoma. However, the impact of hemizygous deletion of CDKN2A/B is unknown. Furthermore, the influence of CDKN2A/B status in IDH-mutant and 1p/19q-codeleted oligodendroglioma remains controversial. We examined the impact of CDKN2A/B status classification, including hemizygous deletions, on the prognosis of IDH-mutant gliomas. Methods We enrolled 101 adults with IDH-mutant glioma between December 2002 and November 2021. CDKN2A/B deletion was evaluated with multiplex ligation-dependent probe amplification (MLPA). Immunohistochemical analysis of p16/MTAP and promoter methylation analysis with methylation-specific MLPA was performed for cases with CDKN2A/B deletion. Kaplan−Meier plots and Cox proportion hazards model analyses were performed to evaluate the impact on overall (OS) and progression-free survival. Results Of 101 cases, 12 and 4 were classified as hemizygous and homozygous deletion, respectively. Immunohistochemistry revealed p16-negative and MTAP retention in cases with hemizygous deletion, whereas homozygous deletions had p16-negative and MTAP loss. In astrocytoma, OS was shorter in the order of homozygous deletion, hemizygous deletion, and copy-neutral groups (median OS: 38.5, 59.5, and 93.1 months, respectively). Multivariate analysis revealed hazard ratios of 9.30 (p=0.0191) and 2.44 (p=0.0943) for homozygous and hemizygous deletions, respectively. Conclusions CDKN2A/B hemizygous deletions exerted a negative impact on OS in astrocytoma. Immunohistochemistry of p16/MTAP can be utilized to validate hemizygous or homozygous deletions in combination with conventional molecular diagnosis.

Autosomal recessive leber hereditary optic neuropathy in a choroideremia carrier. A case report.

Leber hereditary optic neuropathy (LHON) is an inherited progressive optic neuropathy usually caused by mitochondrial DNA mutations. Recently, autosomal recessive (arLHON), which is caused by biallelic mutations in the DNAJC30 gene (usually c.152A > G), has been described. The onset of LHON before the age of 12 is uncommon and it is typically associated with a more variable clinical course and a more favorable visual prognosis than adult-onset LHON. Detailed clinical findings of a female child with vision loss due to arLHON together with choroideremia (CHM) carrier state are presented. Genetic testing for the three most common mitochondrial LHON pathogenic variants was negative. On suspicion of arLHON, genetic testing was continued with the next-generation sequencing (NGS) of the nuclear DNA, identifying a homozygous pathogenic variant in DNAJC3°c.152A > G, p.(Tyr51Cys), but no alterations in the CHM gene. Idebenone treatment was started 4.5 months after the first evaluation. Clinical diagnosis of the CHM carrier state was confirmed by multiplex ligation-dependent probe amplification (MLPA) assay, which revealed a heterozygous deletion of all exons of the CHM. In children with acute or subacute, simultaneous, or sequential vision loss that is unresponsive to immunomodulatory treatment, LHON should be considered as a possible diagnosis. Our case emphasizes the diagnostic advantage of sequencing DNAJC30 in parallel with the mitochondrial DNA, especially in Eastern European descent patients. Genomic rearrangement testing should be considered for patients with a CHM carrier phenotype who have negative results on sequencing tests.

Comprehensive genomic filtering algorithm to expose the cause of skewed X chromosome inactivation. The proof of concept in female haemophilia expression

BackgroundExploring the expression of X linked disorders like haemophilia A (HA) in females involves understanding the balance achieved through X chromosome inactivation (XCI). Skewed XCI (SXCI) may be involved in...

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

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

Next generation sequencing (NGS) and multiplex-ligation dependent probe amplification (MLPA) – Complementary methods in molecular diagnostics of lynch syndrome

Next generation sequencing (NGS) and multiplex-ligation dependent probe amplification (MLPA) – Complementary methods in molecular diagnostics of lynch syndrome

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

ObjectivesWhile 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. MethodsA 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. ResultsTwenty-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. ConclusionsThis 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.

Current Diagnostic Approaches in the Genetic Diagnosis of Disorders of Sex Development.

Disorders of sex development (DSD) are a clinically and genetically highly heterogeneous group of congenital disorders. The most accurate and rapid diagnosis may be possible with a complementary multidisciplinary diagnostic approach, including comprehensive clinical, hormonal, and genetic investigations. Rapid and accurate diagnosis of DSD requires urgency in terms of gender selection and management of the case. Despite the genetic tests performed in current daily practice, the genetic cause is still not elucidated in a significant proportion of cases. Karyotype analysis can be used as a standard for sex chromosome identification. In addition, quantitative fluorescent polymerase chain reaction (QF-PCR) or fluorescence in situ hybridization (FISH) analysis can be used for faster and more cost-effective detection of the sex chromosome and SRY gene. Multiplex ligation-dependent probe amplification (MLPA), single-gene sequence analysis, next-generation sequence analysis (NGSA), targeted NGSA, whole-exome sequencing (WES), and whole-genome sequencing (WGS) analyses can be performed according to preliminary diagnoses. Microarray analysis (array comparative genomic hybridization (aCGH) and single nucleotide polymorphism array (SNPa)) should be performed in cases with syndromic findings and if no pathology is detected with other tests. In DSD cases, the use of optical genome mapping and techniques, which will probably be in daily practice in near future, may be considered. In conclusion, the clinical and genetic diagnosis of DSD is difficult, and molecular genetic diagnosis is often not available. This has psychosocial and health implications for patients and their families. New genetic techniques, especially those targeting the whole genome, may provide a better understanding of DSD through the identification of little-known genetic causes. This review focuses on conventional genetic and next-generation genetic techniques used in the genetic diagnosis of DSD, as well as possible genetic diagnostic techniques and approaches that may be used in routine practice in near future.

Efficiency of CNV-seq in detecting fetal DMD gene deletion or duplication in prenatal diagnosis

Objective: To evaluate the diagnostic efficiency of copy number variation sequencing (CNV-seq) to detect the deletion or duplication of DMD gene in prenatal diagnosis. Methods: A retrospective analysis was carried out on the CNV-seq results of 34 544 fetuses diagnosed in the First People's Hospital of Yunnan Province from January 2018 to July 2023. A total of 156 cases of fetuses were collected, including Group 1:125 cases with family history of Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD), and Group 2:31 cases with no family history but a DMD gene deletion or duplication was detected unexpectedly by CNV-seq. Multiplex ligation-dependent probe amplification (MLPA) was used as a standard method to detect the deletion or duplication. Consistency test was carried out basing on the results of CNV-seq and MLPA of all 156 cases. Results: Comparing to MLPA, CNV-seq had a coincidence rate of 92.3% (144/156) for DMD gene deletion or duplication, with a sensitivity and positive predictive value of 88.2%, with a specificity and negative predictive value of 94.3%, a missed detection rate of 3.8%, and a Kappa value of 0.839. CNV-seq missed 4 cases with deletions and 2 with duplications due to involved fragments less than 100 Kb, among 20 cases of deletions and 6 cases of duplications detected by MLPA in Group 1. In Group 2, the deletions and duplications detected by CNV-seq were 42% (13/31) and 58% (18/31), respectively, in which the percentage of duplication was higher than that in Group 1. Among those 18 cases with duplications, 3 cases with duplication locating in exon 42~67 were likely pathogenic; while 9 cases with duplication covering the 5' or 3' end of the DMD gene, containing exon 1 or 79 and with only one breakpoint within the gene, along with the last 6 cases with duplications locating at chrX: 32650635_32910000 detected only by CNV-seq, which might be judged as variants of uncertain significance. Conclusions: CNV-seq has a good efficiency to detect fetal DMD gene deletion or duplication in prenatal diagnosis, while a further verification test by MLPA is recommended. The duplications on chrX: 32650635_32910000, 5' or 3' end of DMD gene detected by CNV-seq should be carefully verified and assessed because those variants appear to be nonpathogenic polymorphisms.

The Phenotypic Variability Associated with Hepatocyte Nuclear Factor 1B Genetic Defects Poses Challenges in Both Diagnosis and Therapy.

The evolving landscape of clinical genetics is becoming increasingly relevant in the field of nephrology. HNF1B-associated renal disease presents with a diverse array of renal and extrarenal manifestations, prominently featuring cystic kidney disease and diabetes mellitus. For the genetic analyses, whole exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) were performed. Bioinformatics analysis was performed with Ingenuity Clinical Insights software (Qiagen). The patient's electronic record was utilized after receiving informed consent. In this report, we present seven cases of HNF1B-associated kidney disease, each featuring distinct genetic abnormalities and displaying diverse extrarenal manifestations. Over 12 years, the mean decline in eGFR averaged -2.22 ± 0.7 mL/min/1.73 m2. Diabetes mellitus was present in five patients, kidney dysplastic lesions in six patients, pancreatic dysplasia, hypomagnesemia and abnormal liver function tests in three patients each. This case series emphasizes the phenotypic variability and the fast decline in kidney function associated with HNF-1B-related disease. Additionally, it underscores that complex clinical presentations may have a retrospectively straightforward explanation through the use of diverse genetic analytical tools.

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

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