Aminoacylase 1 deficiency: case report on three affected siblings.

Background: Aminoacylase 1 (ACY1, EC 3.5.1.14) deficiency (ACY1D) is a very rare inherited metabolic disease (IMD) with autosomal recessive inheritance (OMIM #609924). Up to date, only 15 cases have been reported in the literature. It is diagnosed by detecting acetylated amino acids among the patient's urine organic acids by gas chromatography-mass spectrometry. Its clinical manifestations are highly variable, ranging from severe neurological symptoms to being asymptomatic. Case Description: We present a 14-year-old boy with mild intellectual disability, speech sound disorder and non-alcoholic fatty liver disease who exhibited increased urinary excretion of N-acetylalanine, N-acetylmethionine and N-acetylglutamine during testing for inherited metabolic disorders. A suspected ACY1D was subsequently confirmed by targeted next generation sequencing, which revealed the presence of a homozygous pathogenic missense mutation in the ACY1 gene, c.1057C>T (p.Arg353Cys). The proband underwent speech education with good outcome. The same homozygous mutation in ACY1 gene was found in the boy's two brothers, who exhibited slightly varied intellectual abilities. Follow-up examinations of the siblings revealed no deterioration in their mental skills. Conclusions: These results suggest that uneven mental abilities in pediatric patients with various disorders including autism spectrum disorder may be sufficient grounds to warrant metabolic testing for ACY1D. The acylglycines urine excretion could be a promising novel metabolic marker for ACY1D testing.

Karyotyping of Lymphocytes and Epithelial Cells of Distinct Embryonic Origin Does Not Help to Predict the Turner Syndrome Features.

BACKGROUND: In Turner syndrome (TS), fluorescent in situ hybridization (FISH) karyotyping offers an alternative to classical karyotyping. OBJECTIVE: We tested the added value of FISH karyotyping from lymphocytes (mesodermal origin), buccal cells (ectodermal origin), and a rear-tongue smear (endodermal origin) to determine the 45,X cell line fraction and its impact on patient phenotype. DESIGN AND PATIENTS: Classical karyotyping and three FISH assays were done in 153 girls and women previously diagnosed with TS in four university hospitals. The 45,X cell line fraction was determined for each method and correlated with the major phenotypic signs. RESULTS: Classical karyotyping identified 45,X/46,XX mosaicism in 77/153 subjects (50%), 45,X monosomy in 52/153 (34%), and other karyotypes in 24/153 (16%). FISH from lymphocytes verified 45,X in 47/52 original cases, whereas 4/52 had 45,X/46,XX and 1/52 45,X/47,XYY mosaicism. The 45,X cell line fraction was higher in FISH from lymphocytes compared to classical karyotyping (median 86.4% vs. 70.0%; p < 0.001), while there was no difference for FISH from buccal or rear-tongue smear cells. The mean 45,X cell line fraction was more abundant in patients with several of the characteristic phenotypic signs compared to patients without them (p < 0.01), but the predictive power was insufficient. CONCLUSION: FISH analysis confirmed the findings of classical karyotyping; only a few 45,X monosomy cases were reclassified as mosaics. The 45,X cell line fraction did not show clinically meaningful prediction of the phenotype. FISH analysis of buccal or rear-tongue epithelial cells may be a non-inferior, less invasive alternative to classical karyotyping.

Ovarian tumors and genetic predisposition.

OBJECTIVE: Summary of knowledge in the field of ovarian cancer and genetic predisposition. RESULTS: Ovarian tumors are usually diagnosed at advanced stages of the disease and the prognosis for these patients is generally poor. The 5-year overall survival rate, regardless of the histopathological type of tumor, is around 44%. Germline mutations causing hereditary tumor syndromes are predominantly involved in the development of epithelial ovarian tumors. The most common is hereditary breast and ovarian cancer syndrome, which is caused by germline mutations in the tumor suppressor genes BRCA1 and BRCA2. Several other tumor suppressor genes and oncogenes are known to be associated with ovarian tumors and cause other types of tumor syndromes. Inherited tumor syndromes include Lynch syndrome, Peutz-Jegers syndrome, Gorlin syndrome, Li-Fraumeni syndrome and others. The indication for genetic examination of germline mutations is given by a clinical geneticist on the basis of the recommendation of the attending physician. At present, every ovarian tumor, primary peritoneal tumor and tube tumor diagnosed at any age is indicated for genetic testing. CONCLUSION: Early identification of genes for hereditary cancer syndromes, thanks to rapidly developing molecular genetic methods, is an important step towards personalized treatment of ovarian cancer and preventive measures in families at risk. It is also important to note that a negative molecular genetic test result does not exclude genetic risk.

Detection of Unknown and Rare Pathogenic Variants in Antithrombin, Protein C and Protein S Deficiency Using High-Throughput Targeted Sequencing.

The deficiency of natural anticoagulants­antithrombin (AT), protein C (PC), and protein S (PS)­is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected­SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels.

Haplotype analysis of the X chromosome in patients with Turner syndrome in order to verify the possible effect of imprinting on selected symptoms.

AIMS: Turner syndrome is the only chromosome monosomy that is postnatally compatible with life. The reported incidence of TS is 1 in 2500 liveborn girls. The phenotype of these girls is highly variable, with cardiac abnormalities being life-threatening defects. The aim of the study was to reveal the possible influence of the parental origin of the X chromosome in these patients on a selected phenotype that is associated with Turner syndrome. Selected symptoms and parameters were: a bicuspid aortic valve, aortic coarctation, lymphoedema, pterygium colli, coeliac disease, thyroiditis, otitis media, diabetes mellitus 2, renal abnormalities, spontaneous puberty, and IVF. METHODS: The X chromosome haplotype was determined for a group of 45,X patients verified by native FISH. A molecular diagnostic method based on the detection of different lengths of X chromosome-linked STR markers using the Argus X-12 QS kit was used to determine the X haplotype. RESULTS: Our results, analysed by Fisher's exact (factorial) test, suggest independence between the maternal/paternal origin of the inherited X chromosome and the presence of the anomalies that were studied (P=1 to P=0.34). CONCLUSION: In the group of 45,X patients, who were precisely selected by means of the native FISH method, no correlation was demonstrated with the parental origin of the X chromosome and the observed symptom.

Searching for genetic variants associated with thrombophilia.

Thrombotic states are inherited or acquired predisposition for thrombosis in the human vascular system. Nowadays Leiden mutation and mutation in prothrombin G20210A contributing to congenital thrombophilia are routinely tested. These mutations have a high prevalence in the population. Congenital deficiencies of protein S, protein C and antithrombin III are rare thrombophilia with lower population frequency, but higher risk of thromboembolic event. The genetic causes are mutations in the genes, which encode these proteins. The choice of proper molecular genetic testing depends on the difference in the detection of well-known single nucleotide polymorphism or unknown/rare variant. For the detection of causative variant FV Leiden and prothrombin G20210A are mostly used PCR-RFLP, reverse Strip Assay®, allele-specific PCR, TaqMan real-time PCR and SNaPshot®. Precise patient selection should precede the genetic testing of rare variants in anticoagulant proteins. It is appropriate to use methodology of massive parallel sequencing supplemented by a methodology for the detection of larger gene rearrangements - MLPA. We are successfully employing this approach in our institute. This methodology is faster with larger analytic capacity compared to commonly used direct sequencing by Sanger method.