Molecular and Functional Characterisation of a Novel Intragenic 12q24.21 Deletion Resulting in MED13L Haploinsufficiency Syndrome.

Background and Objectives: Heterozygous pathogenic variants in the MED13L gene cause impaired intellectual development and distinctive facial features with or without cardiac defects (MIM #616789). This complex neurodevelopmental disorder is characterised by various phenotypic features, including plagiocephaly, strabismus, clubfoot, poor speech, and developmental delay. The aim of this study was to evaluate the clinical significance and consequences of a novel heterozygous intragenic MED13L deletion in a proband with clinical features of a MED13L-related disorder through extensive clinical, molecular, and functional characterisation. Materials and Methods: Combined comparative genomic hybridisation and single-nucleotide polymorphism array (SNP-CGH) was used to identify the changes in the proband's gDNA sequence (DECIPHER #430183). Intragenic MED13L deletion was specified via quantitative polymerase chain reaction (qPCR) and Sanger sequencing of the proband's cDNA sample. Western blot and bioinformatics analyses were used to investigate the consequences of this copy number variant (CNV) at the protein level. CRISPR-Cas9 technology was used for a MED13L-gene-silencing experiment in a culture of the control individual's skin fibroblasts. After the MED13L-gene-editing experiment, subsequent functional fibroblast culture analyses were performed. Results: The analysis of the proband's cDNA sample allowed for specifying the regions of the breakpoints and identifying the heterozygous deletion that spanned exons 3 to 10 of MED13L, which has not been reported previously. In silico, the deletion was predicted to result in a truncated protein NP_056150.1:p.(Val104Glyfs*5), partly altering the Med13_N domain and losing the MedPIWI and Med13_C domains. After MED13L gene editing was performed, reduced cell viability; an accelerated aging process; and inhibition of the RB1, E2F1, and CCNC gene expression were found to exist. Conclusions: Based on these findings, heterozygous intragenic 12q24.21 deletion in the affected individual resulted in MED13L haploinsufficiency due to the premature termination of protein translation, therefore leading to MED13L haploinsufficiency syndrome.

PIGN-Related Disease in Two Lithuanian Families: A Report of Two Novel Pathogenic Variants, Molecular and Clinical Characterisation.

Background and Objectives: Pathogenic variants of PIGN are a known cause of multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1). Many affected individuals have clinical features overlapping with Fryns syndrome and are mainly characterised by developmental delay, congenital anomalies, hypotonia, seizures, and specific minor facial anomalies. This study investigates the clinical and molecular data of three individuals from two unrelated families, the clinical features of which were consistent with a diagnosis of MCAHS1. Materials and Methods: Next-generation sequencing (NGS) technology was used to identify the changes in the DNA sequence. Sanger sequencing of gDNA of probands and their parents was used for validation and segregation analysis. Bioinformatics tools were used to investigate the consequences of pathogenic or likely pathogenic PIGN variants at the protein sequence and structure level. Results: The analysis of NGS data and segregation analysis revealed a compound heterozygous NM_176787.5:c.[1942G>T];[1247_1251del] PIGN genotype in family 1 and NG_033144.1(NM_176787.5):c.[932T>G];[1674+1G>C] PIGN genotype in family 2. In silico, c.1942G>T (p.(Glu648Ter)), c.1247_1251del (p.(Glu416GlyfsTer22)), and c.1674+1G>C (p.(Glu525AspfsTer68)) variants are predicted to result in a premature termination codon that leads to truncated and functionally disrupted protein causing the phenotype of MCAHS1 in the affected individuals. Conclusions: PIGN-related disease represents a wide spectrum of phenotypic features, making clinical diagnosis inaccurate and complicated. The genetic testing of every individual with this phenotype provides new insights into the origin and development of the disease.

Donor Splice Site Variant in SLC9A6 Causes Christianson Syndrome in a Lithuanian Family: A Case Report.

Background and Objectives: The pathogenic variants of SLC9A6 are a known cause of a rare, X-linked neurological disorder called Christianson syndrome (CS). The main characteristics of CS are developmental delay, intellectual disability, and neurological findings. This study investigated the genetic basis and explored the molecular changes that led to CS in two male siblings presenting with intellectual disability, epilepsy, behavioural problems, gastrointestinal dysfunction, poor height, and weight gain. Materials and Methods: Next-generation sequencing of a tetrad was applied to identify the DNA changes and Sanger sequencing of proband's cDNA was used to evaluate the impact of a splice site variant on mRNA structure. Bioinformatical tools were used to investigate SLC9A6 protein structure changes. Results: Sequencing and bioinformatical analysis revealed a novel donor splice site variant (NC_000023.11(NM_001042537.1):c.899 + 1G > A) that leads to a frameshift and a premature stop codon. Protein structure modelling showed that the truncated protein is unlikely to form any functionally relevant SLC9A6 dimers. Conclusions: Molecular and bioinformatical analysis revealed the impact of a novel donor splice site variant in the SLC9A6 gene that leads to truncated and functionally disrupted protein causing the phenotype of CS in the affected individuals.

Compound heterozygous c.598_612del and c.1746-20C > G CAPN3 genotype cause autosomal recessive limb-girdle muscular dystrophy-1: a case report.

BACKGROUND: Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1), also known as calpainopathy, is a genetically heterogeneous disorder characterised by progression of muscle weakness. Homozygous or compound heterozygous variants in the CAPN3 gene are known genetic causes of this condition. The aim of this study was to confirm the molecular consequences of the CAPN3 variant NG_008660.1(NM_000070.3):c.1746-20C > G of an individual with suspected LGMDR1 by extensive complementary DNA (cDNA) analysis. CASE PRESENTATION: In the present study, we report on a male with proximal muscular weakness in his lower limbs. Compound heterozygous NM_000070.3:c.598_612del and NG_008660.1(NM_000070.3):c.1746-20C > G genotype was detected on the CAPN3 gene by targeted next-generation sequencing (NGS). To confirm the pathogenicity of the variant c.1746-20C > G, we conducted genetic analysis based on Sanger sequencing of the proband's cDNA sample. The results revealed that this splicing variant disrupts the original 3' splice site on intron 13, thus leading to the skipping of the DNA fragment involving exon 14 and possibly exon 15. However, the lack of exon 15 in the CAPN3 isoforms present in a blood sample was explained by cell-specific alternative splicing rather than an aberrant splicing mechanism. In silico the c.1746-20C > G splicing variant consequently resulted in frameshift and formation of a premature termination codon (NP_000061.1:p.(Glu582Aspfs*62)). CONCLUSIONS: Based on the results of our study and the literature we reviewed, both c.598_612del and c.1746-20C > G variants are pathogenic and together cause LGMDR1. Therefore, extensive mRNA and/or cDNA analysis of splicing variants is critical to understand the pathogenesis of the disease.

A de novo 13q31.3 microduplication encompassing the miR-17 ~ 92 cluster results in features mirroring those associated with Feingold syndrome 2.

Hemizygosity of the MIR17HG gene encoding the miR-17 ~ 92 cluster is associated with Feingold syndrome 2 characterized by intellectual disability, skeletal abnormalities, short stature, and microcephaly. Here, we report on a female with a de novo 13q31.3 microduplication encompassing MIR17HG but excluding GPC5. She presented developmental delay, skeletal and digital abnormalities, and features such as tall stature and macrocephaly mirroring those of Feingold syndrome 2 patients. The limited extent of the proband's rearrangement to the miR cluster and the corresponding normal expression level of the neighboring GPC5 in her cells, together with previously described data on affected individuals of two families carrying overlapping duplications of the miR-17 ~ 92 cluster that comprise part of GPC5, who likewise presented macrocephaly, developmental delay, as well as skeletal, digital and stature abnormalities, allow to define a new syndrome due to independent microduplication of the miR-17 ~ 92 cluster.

De novo splice site variant of ARID1B associated with pathogenesis of Coffin-Siris syndrome.

BACKGROUND: Coffin-Siris syndrome is an extremely rare syndrome associated with developmental and congenital anomalies. It is caused by heterozygous pathogenic variants of ARID1A, ARID1B, SMARCA4, SMARCB1, SMARCE1, and SOX11. METHODS: This case study presents the whole exome sequencing of a patient with characteristic clinical features of Coffin-Siris syndrome. Analysis included Sanger sequencing of complementary DNA and bioinformatic analysis of the variant. RESULTS: Analysis of cDNA Sanger sequencing data revealed that the donor splice site variant led to skipping of exon 19. Further, bioinformatic analysis predicted abnormal splicing in a translational frameshift of 11 amino acids and the creation of a premature termination codon. Results found a novel de novo splice site variant c.5025+2T>C in the ARID1B and truncated 1 633 amino acid protein NP_065783.3:p. (Thr1633Valfs*11). CONCLUSION: Truncated ARID1B resulted in loss of the BAF250 domain, which is part of SWI/SNF-like ATP-dependent chromatin remodeling complex. The severe clinical manifestation presented by the proband was attributed to the disappearance of the BAF250 domain in the ARID1B protein. Our finding provides strong evidence that this pathogenic variant of exon 19 caused a frameshift mutation in the ARID1B at the terminal exon, resulting in the expression of a severe phenotype of CSS.

A novel CHD7 variant disrupting acceptor splice site in a patient with mild features of CHARGE syndrome: a case report.

BACKGROUND: CHARGE syndrome (MIM# 214800)-which is characterised by a number of congenital anomalies including coloboma, ear anomalies, deafness, facial anomalies, heart defects, atresia choanae, genital hypoplasia, growth retardation, and developmental delay-is caused by a heterozygous variant in the CHD7 (MIM# 608892) gene located on chromosome 8q12. We report the identification of a novel c.5535-1G > A variant in CHD7 and provide the evaluation of its effect on pre-mRNA splicing. CASE PRESENTATION: In this study, we report on a female presenting features of CHARGE syndrome. A novel heterozygous CHD7 variant c.5535-1G > A located in the acceptor splice site of intron 26 was identified in the proband's DNA sample after analysis of whole exome sequencing data. In silico predictions indicating that the variant is probably pathogenic by affecting pre-mRNA splicing were verified by genetic analysis based on reverse transcription of the patient's RNA followed by PCR amplifications performed on synthesised cDNA and Sanger sequencing. Sanger sequencing of cDNA revealed that the c.5535-1G > A variant disrupts the original acceptor splice site and activates a cryptic splice site only one nucleotide downstream of the pathogenic variant site. This change causes the omission of the first nucleotide of exon 27, leading to a frameshift in the mRNA of the CHD7 gene. Our results suggest that the alteration induces the premature truncation of the CHD7 protein (UniProtKB: Q9P2D1), thus resulting in CHARGE syndrome. CONCLUSION: Genetic analysis of novel splice site variant underlines its importance for studying the pathogenic splicing mechanism as well as for confirming a diagnosis.

Novel GLI3 variant causes Greig cephalopolysyndactyly syndrome in three generations of a Lithuanian family.

BACKGROUND: Preaxial polydactyly type IV, also referred as polysyndactyly, has been described in a few syndromes. We present three generations of a family with preaxial polydactyly type IV and other clinical features of Greig cephalopolysyndactyly syndrome (GCPS). METHODS AND RESULTS: Sequencing analysis of the GLI3 coding region identified a novel donor splice site variant NC_000007.14(NM_000168.6):c.473+3A>T in the proband and the same pathogenic variant was subsequently identified in other affected family members. Functional analysis based on Sanger sequencing of the proband's complementary DNA (cDNA) sample revealed that the splice site variant c.473+3A>T disrupts the original donor splice site, thus leading to exon 4 skipping. Based on further in silico analysis, this pathogenic splice site variant consequently results in a truncated protein NP_000159.3:p.(His123Argfs*57), which lacks almost all functionally important domains. Therefore, functional cDNA analysis confirmed that the haploinsufficiency of the GLI3 is the cause of GCPS in the affected family members. CONCLUSION: Despite the evidence provided, pathogenic variants in the GLI3 do not always definitely correlate with syndromic or nonsyndromic clinical phenotypes associated with this gene. For this reason, further transcriptomic and proteomic evaluation could be suggested.