[Identification of a Family with SUFU Germline Deletion Based on a Case of Desmoplastic Medulloblastoma in an Infant]. / Identifikace rodiny s nosicstvím germinální delece genu SUFU na podklade dia-gnózy desmoplastického meduloblastomu u batolete.

BACKGROUND: Medulloblastoma, an embryonal neuroectodermal tumor of the cerebellum, is the most common malignant brain tumor in children. There are approximately 15 cases diagnosed in the Czech Republic each year. The recent World Health Organization classification recognizes several histopathological subtypes of medulloblastoma: classical, desmoplastic/ nodular with its extensive-nodularity variant, and anaplastic/ large-cell variant. Further molecular analysis identified four basic subgroups of medulloblastoma: WNT, SHH, Group 3, and Group 4. The subgroup of SHH meduloblastoma is associated with somatic mutations of SHH, PTCH1, SUFU, SMO and TP53, while the most common mutations found in infants up to three years of age were PTCH1 and SUFU. The majority of medulloblastomas are sporadic diseases, whereas only about 5- 10% of all cases occur in connection with hereditary genetic syndromes. CASE: We present a case of a 21-months old girl diagnosed with a localized posterior fossa tumor. The histopathological examination revealed a desmoplastic/ nodular medulloblastoma. The treatment comprised a radical exstirpation of the tumor followed by adjuvant chemotherapy. With the use of array-CGH, a partial biallelic deletion of the SUFU gene (locus 10q24.32) was detected in the tumor DNA, whereas a monoallelic deletion was found in the peripheral lymphocyte DNA of the patient. These findings were confirmed by an independent qPCR method. Monoallelic germline deletion of SUFU was also identified in the patients mother, who was a healthy carrier. Pedigree of the family suggested a transition of the germline deletion of SUFU, since another brain tumors (including one case diagnosed before the age of three years) were identified in previous generations. CONCLUSION: Germline mutations in SUFU gene are believed to predispose to infant desmoplastic/ nodular medulloblastomas, basal cell carcinomas and meningiomas. The susceptibility gene shows autosomal dominant inheritance with an incomplete penetrance. There is no evidence-based surveillance strategy suggested for the carriers of germline SUFU mutations/ deletions so far. Our recommendation is based both on a family history of our patient and similar cases described in the literature. Since the germinal mutations in SUFU are responsible for up to 50% of all desmoplastic medulloblastomas in children under three years of age, genetic testing of SUFU should be encouraged in this population of patients.

[Li-Fraumeni syndrome - a proposal of complex prevention care for carriers of TP53 mutation with total-body MRI]. / Li-Fraumeni syndrom - návrh komplexní preventivní péce o nosice TP53 mutace s pouzitím celotelové magnetické rezonance.

Li-Fraumeni syndrome (LFS) is one of the most serious hereditary cancer syndromes with high risk of malignancy already in childhood. Adrenocortical carcinoma, brain tumor, leukemia, sarcoma are the most frequent malignancies in children. Early breast cancer, brain tumor, sarcoma, skin cancer, gastrointestinal, lung, gynecological, hematological and other malignancies can be seen in adults. Predictive testing in families with detected LFS and TP53 mutation is offered from the age of 18 for various reasons. One of the most important reasons is a very limited effectivity of prevention especially in children, also the possible risk of psychological harm to the child and his family caused by the diagnosis of this syndrome. Progress in diagnostic methods, especially total body MRI, enables to propose preventive care for early cancer diagnoses for children and adults. Biochemical tests, ultrasound, MRI may improve survival of these high risk individuals and support the possibility of predictive testing in children.

Unique Combination of 22q11 and 14qter Microdeletion Syndromes Detected Using Oligonucleotide Array-CGH.

We report an infant with a unique combination of 22q11 deletion syndrome and 14q terminal deletion syndrome. The proband had clinical symptoms compatible with diagnosis of 22q11 deletion syndrome: microcephaly, micrognathia, high-arched palate, hypertelorism, short palpebral fissures, square nasal root, prominent tubular nose, hypoplastic nasal alae, bulbous nasal tip, dysplastic low-set ears, short philtrum, and heart defect, but no cell-mediated immunodeficiency typical for the syndrome. G-banding and fluorescence in situ hybridization analyses revealed a karyotype 45,XY,der(14)t(14;22)(q32.3;q11.2),-22.ish del(14)(q32.33)(D14S1420-),del(22)(q11.2q11.2)(N25-). Subsequent analyses disclosed a translocation between chromosomes 14 and 22 in the proband's mother with a deleted 14q telomere. Using comparative genome hybridization on oligonucleotide-based microarray (array-CGH), the deletion at 22q11.21 in the size of ∼4.25 Mb was revealed in the proband as well as the deletion of the telomeric area at 14q32.33qter (∼3.24 Mb) in the proband and his mother. However, both the proband and his mother showed mild symptoms (microcephaly, thin lips, carp-shaped mouth) typical for patients with the described terminal 14q deletion syndrome.

The effect of the swim-up and hyaluronan-binding methods on the frequency of abnormal spermatozoa detected by FISH and SCSA in carriers of balanced chromosomal translocations.

BACKGROUND: The swim-up and hyaluronan (HA)-binding methods are used for the selection of good quality spermatozoa to improve pregnancy rates and embryo quality and to reduce the number of miscarriages after IVF. We evaluated whether the processing of sperm by these methods reduces the frequency of spermatozoa with abnormal karyotypes and altered chromatin quality in balanced translocation carriers. METHODS: Semen samples of 12 carriers of balanced chromosomal translocations were analysed for the frequency of spermatozoa, which are chromosomally unbalanced due to the segregation of balanced translocations, aneuploidies for chromosomes 7, 8, 13, 18, 21, X or Y, diploid sperm or sperm with fragmented DNA and poorly condensed chromatin. Results obtained by fluorescence in situ hybridization (FISH) and sperm chromatin structure assay were compared between ejaculated (n = 12), swim-up (n = 12) and HA-binding processed (n = 6) semen samples of the translocation carriers and with the control group (n = 10). RESULTS: The mean frequencies of unbalanced segregation products were 17.5 and 16.5% in neat and swim-up processed samples from Robertsonian translocation carriers, and 55.4, 54.5 and 50.9% in neat, swim-up and HA-bound sperm samples from reciprocal translocation carriers. Significant decreases in the frequency of sperm showing chromosome 18 and XY disomy and of diploidy, and in the rates of high-density staining sperm were observed in the motile swim-up fractions. There were significantly more sperm showing fragmented chromatin in the group of translocation carriers than in the control group, but no differences in the aneuploidy and diploidy rates were observed. CONCLUSIONS: The swim-up method is suitable for selection of sperm with condensed chromatin and a lower frequency of some aneuploidies and of diploidy. The frequency of spermatozoa chromosomally unbalanced due to the segregation of reciprocal (but not Robertsonian) translocations is significantly lower in HA-bound sperm. However, the advantages of either method for selecting normal sperm are limited.

SHOX gene defects and selected dysmorphic signs in patients of idiopathic short stature and Léri-Weill dyschondrosteosis.

The aim of the study was to analyze frequency of SHOX gene defects and selected dysmorphic signs in patients of both idiopathic short stature (ISS) and Léri-Weill dyschondrosteosis (LWD), all derived from the Czech population. Overall, 98 subjects were analyzed in the study. Inclusion criteria were the presence of short stature (-2.0 SD), in combination with at least one of the selected dysmorphic signs for the ISS+ group; and the presence of Madelung deformity, without positive karyotyping for the LWD+ group. Each proband was analyzed by use of P018 MLPA kit, which covers SHOX and its regulatory sequences. Additionally, mutational analysis was done of the coding portions of the SHOX. Both extent and breakpoint localizations in the deletions/duplications found were quite variable. Some PAR1 rearrangements were detected, without obvious phenotypic association. In the ISS+ group, MLPA analysis detected four PAR1 deletions associated with a SHOX gene defect, PAR1 duplication with an ambiguous effect, and two SHOX mutations (13.7%). In the LWD+ group, MLPA analysis detected nine deletions in PAR1 region, with a deleterious effect on SHOX, first reported case of isolated SHOX enhancer duplication, and SHOX mutation (68.8%). In both ISS+ and LWD+ groups were positivity associated with a disproportionately short stature; in the ISS+ group, in combination with muscular hypertrophy. It seems that small PAR1 rearrangements might be quite frequent in the population. Our study suggests disproportionateness, especially in combination with muscular hypertrophy, as relevant indicators of ISS to be the effect of SHOX defect.

Analysis of chromosomal aberrations in patients with mental retardation using the array-CGH technique: a single Czech centre experience.

Submicroscopic structural chromosomal aberrations (microduplications and microdeletions) are believed to be common causes of mental retardation. These so-called copy number variations can now be routinely detected using various platforms for array-based comparative genomic hybridization (array-CGH), which allow genome-wide identification of pathogenic genomic imbalances. In this study, oligonucleotide-based array-CGH was used to investigate a panel of 23 patients with mental retardation and developmental delay, dysmorphic features or congenital anomalies. Array-CGH confirmed or revealed 16 chromosomal aberrations in a total of 12 patients. Analysis of parental samples showed that five aberrations had occurred de novo: del(1)(p36.33p36.23), del(4)(p16.3p16.2) joined with dup(8)(p23.3p23.1), del(6)(q14.1q15), del(11)(q13.1q13.4). Three aberrations appeared to be inherited from an unaffected parent: dup(3)(q29), del(6)(q12), dup(16)(p13.11). Six aberrations appeared to be inherited from a parental carrier: del(1)(p36.33) joined with dup(12)(q24.32), del(21)(q22.2q22.3) joined with dup(11)(q24.2q25), del(X)(q22.3) and del(1)(q21.1). In two cases, parents were not available for testing: del(17)(q11.2q12) and del(2)(q24.3q31.1). Our results show that the use of oligonucleotide-based array- CGH in a clinical diagnostic laboratory increases the detection rate of pathogenic submicroscopic chromosomal aberrations in patients with mental retardation and congenital abnormalities, but it also presents challenges for clinical interpretation of the results (i.e., distinguishing between pathogenic and benign variants). Difficulties with analysis notwithstanding, the array-CGH is shown to be a sensitive, fast and reliable method for genome-wide screening of chromosomal aberrations in patients with mental retardation and congenital abnormalities.

Mutation analysis of candidate genes SCN1B, KCND3 and ANK2 in patients with clinical diagnosis of long QT syndrome.

The long QT syndrome (LQTS) is a monogenic disorder characterized by prolongation of the QT interval on electrocardiogram and syncope or sudden death caused by polymorphic ventricular tachycardia (torsades de pointes). In general, mutations in cardiac ion channel genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) have been identified as a cause for LQTS. About 50-60 % of LQTS patients have an identifiable LQTS causing mutation in one of mentioned genes. In a group of 12 LQTS patients with no identified mutations in these genes we have tested a hypothesis that other candidate genes could be involved in LQTS pathophysiology. SCN1B and KCND3 genes encode ion channel proteins, ANK2 gene encodes cytoskeletal protein interacting with ion channels. To screen coding regions of genes SCN1B, KCND3, and 10 exons of ANK2 following methods were used: PCR, SSCP, and DNA sequencing. Five polymorphisms were found in screened candidate genes, 2 polymorphisms in KCND3 and 3 in SCN1B. None of found polymorphisms has coding effect nor is located close to splice sites or has any similarity to known splicing enhancer motifs. Polymorphism G246T in SCN1B is a novel one. No mutation directly causing LQTS was found. Molecular mechanism of LQTS genesis in these patients remains unclear.

Charcot-Marie-Tooth neuropathy type 1A combined with Duchenne muscular dystrophy.

We report a 24-year-old male with an unusual combination of two inherited neuromuscular disorders--Charcot-Marie-Tooth (CMT) disease type 1A and Duchenne muscular dystrophy (DMD). A phenotypic presentation of this patient included features of both these disorders. Nerve conduction studies revealed demyelinating peripheral neuropathy. Electromyography showed a profound myogenic pattern. The serum creatine kinase level was highly elevated. Muscle biopsy revealed a dystrophic picture with deficient dystrophin immunostaining. CMT1A duplication on chromosome 17p11.2 was found. The frame-shift mutation c.3609-3612delTAAAinsCTT (p.K1204LfsX11) was detected in the dystrophin gene by analysing mRNA isolated from the muscle tissue. The patient inherited both these mutations from his mother. The combination of CMT1A and DMD has not been reported as yet.

[Mutational analysis of LQT genes in individuals with drug induced QT interval prolongation]. / Mutacní analáza LQT genu u jedincu s polékovým prodlouzením QT intervalu.

BACKGROUND: In a long list of non-cardiovascular drugs a risk of QT interval prolongation and thus an increased risk of malignant arrhythmias has been described. The precise mechanism remains unclear. Many of these drugs are potent blockers of cardiac ion channels. Thus, prolongation of repolarization could be caused by latent ion channel genes mutations which are revealed under stress conditions. GROUP OF PATIENTS AND METHODS: Patients were recruited in screening of antipsychotic drugs with proarrhythmic potential, another sporadic cases were reffered from regional hospitals. In 13 individuals pathologic values of corrected QT interval (> 0.44 s in males, > 0.46 s in females) were observed. Eleven patients gave their consent to mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2 and KCNJ2 genes (associated with congenital long QT syndrome). RESULTS: At present complete results of mutational analysis are available in 8 patients. In 5 individuals changes in DNA sequence were found which are considered normal variants according to the literature (nucleotide and aminoacid polymorphisms, intronic variants). In 1 male a KCNQ1 gene mutation A590T was identified (yet not reported in literature). CONCLUSION: Mechanisms of drug-induced QT interval prolongation is complex and it cannot be explained simply by ion channel disorders.

[The role of ergometric testing in diagnosis of latent long QT syndrome]. / Postavení ergometrického vysetrení v diagnostice latentního syndromu dlouhého QT intervalu.

BACKGROUND: The long QT syndrome is a genetically determined disease based on mutations of ion membrane channel genes. The resulting prolongation of repolarization increases a risk of malignant ventricular arrhythmias and sudden death. The diagnosis is problematic in individuals with borderline or even normal corrected QT interval value. Ventricular arrhythmias in this syndrome are often provoked by exercise, therefore exercise testing is considered as a useful differential diagnostic method. METHODS: In a 24-member family an occurrence of the long QT syndrome was established clinically in 7 patients. In other 3 members borderline corrected QT interval values were found (0.44-0.46 s). In these individuals a stress testing by bicycle ergometry was performed. RESULTS: During exercise in a 28-year old man (patient III/6) the corrected QT interval prolonged to 0.53 s, in a 27-year old man (patient III/2) and in a 20-year old woman (patient III/9) physiological QT interval shortenings were observed (0.4 s during peak exercise). The diagnosis was confirmed by molecular genetic investigation. In patient III/6 (along with other symptomatic family members) a mutation in exon 7 of KCNQ1 (previously called KVLQT1) gene was found. In patients III/2 and III/9 no signs of KCNQ1 gene pathology were present. CONCLUSIONS: In families with clinically established diagnosis of long QT syndrome occurrence the exercise testing reveals previously asymptomatic individuals, at least in LQT1 type (which is the most common). In general stress testing and measurement of QT interval dynamics is a necessary part of arrhythmological investigation especially in young individuals with history of syncope.

Agammaglobulinaemia in a girl with a mosaic of ring 18 chromosome.

A patient with a mosaic karyotype 45,XX,-18/46,XX,dic r(18)/46,XX,r(18) with multiple phenotypic abnormalities and immunodeficiency was presented at the age of 14 years. Immunological investigation revealed markedly decreased IgG, IgA and in two of three evaluations also IgM levels. Although selective IgA deficiency is frequent in patients with a ring chromosome 18, this is the third patient described with decreased levels of other immunoglobulin isotypes. The association of chromosome 18 partial deletions and immunoglobulin abnormalities suggests the presence of an as yet unrecognised gene with a pivotal role for immunoglobulin production on chromosome 18.

DNA diagnosis and clinical manifestations of autosomal dominant polycystic kidney disease.

At least 2 genes, detectable by DNA methods, encode autosomal dominant polycystic kidney disease (ADPKD), which remains the most frequent and serious hereditary renal disease. PKD1 gene, localized on chromosome 16, responds for the clinical course in the majority of ADPKD patients, whereas PKD2 gene, localized on chromosome 4, is responsible for less than 10-15% of cases, with presumed milder phenotypic manifestations. To start the clinical and genetic correlation in patients with different genotypes (PKD1 vs. PKD2) in the Czech population, a pilot group of 88 patients with ADPKD was analysed. Families with PKD1 (n = 44) represented 95.6% and families with PKD2 (n = 2) 4.4% of all families investigated (n = 46). Our clinical analysis, yet based only on a limited number of PKD2 subjects, does not definitely support the concept of a milder phenotype and prognosis in PKD2 versus PKD1 patients, in terms of mean age of diagnosis (29 vs. 29 years), mean age at onset of arterial hypertension (33 vs. 33 years), more favourable renal function or ultrasound findings.