A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Novel genetic variant of HPS1 gene in Hermansky-Pudlak syndrome with fulminant progression of pulmonary fibrosis: a case report.

BACKGROUND: Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder that is associated with oculocutaneous albinism, bleeding diathesis, granulomatous colitis, and highly penetrant pulmonary fibrosis in some subtypes. Homozygous or compound heterozygous pathological variants in HPS1, HPS3, HPS4, and several other genes lead to clinical manifestation of the disease. CASE PRESENTATION: A 57-year-old female was admitted with congenital oculocutaneous albinism, thrombocytopathy and late-onset accelerated pulmonary fibrosis (first symptoms from age 50 onwards). Chest high-resolution computed tomography identified thickening of peribronchovascular interstitium, bronchiectasis, reticulations, honeycombing, ground glass opacities and lung parenchyma consolidations. HPS was clinically suspected. We performed whole exome sequencing (WES), a form of massive parallel sequencing, of proband-parents trio. Whole exome libraries were processed using KAPA Hyper Prep Kit, SeqCap EZ MedExome Enrichment Kit and HyperCap Bead Kit according to the SeqCap EZ HyperCap Workflow. The paired-end 2 × 75 bp sequencing was performed on the Illumina NextSeq 500 Sequencer (Illumina Inc., USA). Furthermore, obtained variants by WES were evaluated using a virtual panel of genes: HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, and PLDN. We identified a compound heterozygous genotype in HPS1 gene in the proband. We identified a pathogenic frameshift variant c.1189delC; p.(Gln397Serfs*2), resulting in a premature stop codon. This variant has been previously associated with HPS. Furthermore, we characterized previously undescribed nonsense variant c.1507C > T; p.(Gln503*), resulting in a premature stop codon and mRNA degradation through nonsense-mediated decay. Sanger sequencing validated the presence of both variants and simultaneously confirmed the heterozygous carrier status of parents. Unfortunately, the patient died due to fulminant progression of pulmonary fibrosis 2 months after diagnostics. CONCLUSIONS: Compound heterozygous mutations in HPS1 in the proband lead to disruption of HPS1 gene and clinical manifestation of HPS with severe pulmonary fibrosis. This case illustrates the need to consider HPS in differential diagnostics of pulmonary fibrosis. Pulmonary fibrosis is a common cause of death in HPS patients. Earlier diagnosis may enable better treatment for these patients.

Germline variant of CTC1 gene in a patient with pulmonary fibrosis and myelodysplastic syndrome.

Introduction: Telomeropathies are associated with a wide range of diseases and less common combinations of various pulmonary and extrapulmonary disorders. Case presentation: In proband with high-risk myelodysplastic syndrome and interstitial pulmonary fibrosis, whole exome sequencing revealed a germline heterozygous variant of CTC1 gene (c.1360delG). This "frameshift" variant results in a premature stop codon and is classified as likely pathogenic/pathogenic. So far, this gene variant has been described in a heterozygous state in adult patients with hematological diseases such as idiopathic aplastic anemia or paroxysmal nocturnal hemoglobinuria, but also in interstitial pulmonary fibrosis. Described CTC1 gene variant affects telomere length and leads to telomeropathies. Conclusions: In our case report, we describe a rare case of coincidence of pulmonary fibrosis and hematological malignancy caused by a germline gene mutation in CTC1. Lung diseases and hematologic malignancies associated with short telomeres do not respond well to standard treatment.

Extensive, 3.8 Mb-Sized Deletion of 22q12 in a Patient with Bilateral Schwannoma, Intellectual Disability, Sensorineural Hearing Loss, and Epilepsy.

Introduction: In contrast with the well-known and described deletion of the 22q11 chromosome region responsible for DiGeorge syndrome, 22q12 deletions are much rarer. Only a few dozen cases have been reported so far. This region contains genes responsible for cell cycle control, chromatin modification, transmembrane signaling, cell adhesion, and neural development, as well as several cancer predisposition genes. Case Presentation: We present a patient with cleft palate, sensorineural hearing loss, vestibular dysfunction, epilepsy, mild to moderate intellectual disability, divergent strabism, pes equinovarus, platyspondylia, and bilateral schwannoma. Using Microarray-based Comparative Genomic Hybridization (aCGH), we identified the de novo 3.8 Mb interstitial deletion at 22q12.1→22q12.3. We confirmed deletion of the critical NF2 region by MLPA analysis. Discussion: Large 22q12 deletion in the proband encases the critical NF2 region, responsible for development of bilateral schwannoma. We compared the phenotype of the patient with previously reported cases. Interestingly, our patient developed cleft palate even without deletion of the MN1 gene, deemed responsible in previous studies. We also strongly suspect the DEPDC5 gene deletion to be responsible for seizures, consistent with previously reported cases.

A patient with Smith-Lemli-Opitz syndrome: novel mutation of the DHCR7 gene and effects of therapy with simvastatin and cholesterol supplement.

BACKGROUND: The Smith-Lemli-Opitz (SLO) syndrome is a multiple congenital anomaly with mental retardation due to a decreased or lack of activity of 7-dehydrocholesterol reductase as a consequence of mutations of the DHCR7 gene. This paper describes a special patient with SLO syndrome. Laboratory examination showed low cholesterol (2.77 mmol/L) and increased 7-dehydrocholesterol level (102 mg/L). Molecular genetic analysis revealed a compound heterozygosity c.964-1G>C/p.G366V (c.G1370T) of the proband. The p.G366V is a novel mutation of the DHCR7 gene with guanine by thymine nucleotide exchange resulting in glycin by valin amino acid exchange in the dehydrocholesterol reductase enzyme. Simvastatin (0.2 mg/kg/day) and cholesterol replacement therapy (150-250 mg/kg/day) led to significant improvement in the patient's laboratory findings (7-dehydrocholesterol, cholesterol) as well as in his behavior and gross motor function. CONCLUSION: Our patient demonstrates that the c.964-1G>C/p.G366V (c.G1370T) genotype of combined heterozygosity is associated with a typical form of SLO syndrome along with moderately altered laboratory findings and a favorable biochemical response to cholesterol and simvastatin treatment.

Diagnosis of Bloom Syndrome in a Patient with Short Stature, Recurrence of Malignant Lymphoma, and Consanguineous Origin.

Bloom syndrome is an autosomal recessive disorder characterized by prenatal and postnatal growth deficiency, photosensitive skin changes, immune deficiency, insulin resistance, and a greatly increased risk of early-onset cancer and development of multiple malignancies. Loss-of-function variants of the BLM gene, which codes for a RecQ helicase, cause Bloom syndrome. We report a consanguineous family, with 2 siblings showing clinical signs of suspected chromosome breakage disorder. One of them developed recurrent malignant lymphoma during lifetime. We performed next-generation sequencing analysis, focusing on cancer predisposition syndromes. We identified a homozygous pathogenic nonsense variant c.1642C>T (p.Gln548*) in the BLM gene in the proband, associated with Bloom syndrome. Sanger sequencing validated the presence of a homozygous pathogenic variant in the proband and also in the brother with short stature. In this article, we will focus on the clinical presentation of the syndrome in this particular family as well as the characteristics of malignancies found in the proband.

MicroRNA-210 expression during childbirth and postpartum as a potential biomarker of acute fetal hypoxia.

OBJECTIVE: To explore whether miR-210 expression can be used as a diagnostic and prognostic marker in acute fetal hypoxia. METHODS: Whole blood samples of 29 women and their fetuses without hypoxia and 24 women and their fetuses with hypoxia were analysed in this study. Reverse transcription and quantitative real-time PCR were used to measure the expression of miR-210. Expression level differences between the control and hypoxic group in labour time and postpartum change fold were analyzed by standard statistical tests. RESULTS: We confirmed that miR-210 is significantly more upregulated in fetal blood with acute hypoxia when compared to maternal blood (P Conclusions: Our study confirmed miR-210 upregulation in the blood of pregnant women with acute fetal hypoxia at the time of labour compared to pregnant women without acute fetal hypoxia. Additional investigation should be done to determine miR-210 clearance and the possibility of using miR-210 as a diagnostic and prognostic marker.

Complex analysis of the p53 tumor suppressor in lung carcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. The p53 tumor suppressor is a transcription factor controlling expression of its target genes in response to various stress stimuli. Mutations of the TP53 gene occur very frequently in lung carcinomas and they play an important role in both oncogenic transformation of lung epithelial cells and lung carcinoma progression. We determined the TP53 status in 42 samples of squamous cell lung carcinoma (SQCC) and 56 samples of lung adenocarcinoma (AC) by the functional analysis FASAY and its variant called split assay. Altogether, we detected 64 TP53 mutations in 63 patients and analyzed them by cDNA and gDNA sequencing. The TP53 mutations were found in 76.2% (32/42) of SQCC cases, and 55.4% (31/56) of ACs. Immunoblotting revealed the p53 protein accumulation in 18 samples (42.9%) among SQCC cases and 19 samples (33.9%) among AC cases. Using fluorescence in situ hybridization we detected loss of the TP53-specific 17p13.3 locus in 23 from 41 analyzed SQCC samples (56.1%) and in 20 from 54 analyzed AC samples (37.0%). We did not find any statistically significant differences in overall and disease-free survival in relation to TP53 status.

Identification and characterization of large deletions in the phenylalanine hydroxylase (PAH) gene by MLPA: evidence for both homologous and non-homologous mechanisms of rearrangement.

Large gene deletions and duplications were analyzed in 59 unrelated phenylketonuria (PKU) patients negative for phenylalanine hydroxylase (PAH) mutations on one or both alleles from previous exon by exon analysis. Using the novel multiplex ligation-dependent probe amplification (MLPA) method, a total of 31 partial PAH deletions involving single exons were identified in 31 PKU patients. Nineteen cases exhibited deletion of exon 5, and 12 cases provided evidence for the deletion of exon 3. Subsequently, using restriction enzyme digestion and DNA sequencing, three different large deletions, EX3del4765 (12 cases), EX5del955 (2 cases) and EX5del4232ins268 (17 cases) were identified and confirmed by long-range PCR and by the analysis of aberrant transcripts. Altogether, the 31 large deletions presented account for 3% of all PAH mutant alleles investigated in Czech PKU patients. Bioinformatic analysis of three breakpoints showed that the mutation EX3del4765 had arisen through an Alu-Alu homologous recombination, whereas two other mutations-the EX5del955 and EX5del4232ins268, had been created by a non-homologous end joining (NHEJ). We conclude that MLPA is a convenient, rapid and reliable method for detection of intragenic deletions in the PAH gene and that a relatively high number of alleles with large deletions are present in the Slavic PKU population.