Gabriella P. Szabó

Mutational Spectrum of Smith-Lemli-Opitz Syndrome Patients in Hungary

Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder characterized by multiple congenital abnormalities and mental retardation. The condition is caused by the deficiency of 7-dehydrocholesterol reductase (DHCR7) which catalyzes the final step in cholesterol biosynthesis. Biochemical diagnosis is based on increased concentration of 7-dehydrocholesterol (7-DHC) in the patient serum. Both life expectancy and quality of life are severely affected by the disease. The estimated prevalence of SLO syndrome ranges between 1:20,000 and 1:40,000 among Caucasians. Although the mutational spectrum of the disease is wide, approximately 10 mutations are responsible for more than 80% of the cases. These mutations show a large interethnic variability. There are no mutation distribution data from Hungary to date. Thirteen patients were diagnosed with SLO syndrome in our laboratory. As first-line tests, serum 7-DHC and total cholesterol were measured and, in positive cases, molecular genetic analysis of the DHCR7 gene was performed. Complete genetic background of the disease could be identified in 12 cases. In 1 case only 1 mutation was detected in a heterozygote form. One patient was homozygous for the common splice site mutation c.964–1G>C, while all other patients were compound heterozygotes. One novel missense mutation, c.374A>G (p.Tyr125Cys) was identified.

Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods

Abstract Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods – PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) – was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%–2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes. Conclusions: This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.

Detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation

A kromoszomak szubtelomerikus regioi genben gazdag teruletek, atrendeződesuk hagyomanyos kromoszomaanalizissel nem detektalhato. Mivel a mentalis retardaciok kozel 7%-aert felelősek, kimutatasuk diagnosztikai szempontbol jelentős, es lehetőseget nyujt az ismetlődes megakadalyozasara is. A kimutatasukra alkalmas modszerek egyike a szubtelomerikus fl uoreszcencia in situ hibridizacio. Otvenkilenc idiopathias mentalisan retardalt beteg kozul 35 kozepes/sulyos ertelmi fogyatekost valasztottunk ki a nemzetkozi irodalomban ajanlott kriteriumok alapjan. Kozuluk 6 beteg eseteben mutattunk ki szubtelomerikus aberraciot, 5 familiaris (ket csalad), egy de novo esetnek bizonyult. Huszonkilenc betegben szubtelomerikus kromoszomaatrendeződest nem igazoltunk. A 6 beteg kozul kettőben 8pter deleciot es 12pter duplikaciot, haromban 21qter deleciot es 10pter duplikaciot azonositottunk kiegyensulyozatlan transzlokacio formajaban. Egy betegnel de novo keletkezett 3qter deleciot detektaltunk. Az elteresek eredetenek tisztazasa soran 12 egeszseges csaladtag kozul ot bizonyult kiegyensulyozott transzlokaciohordozonak. Az irodalmi adatokkal osszhangban megallapitottuk, hogy a fenotipust a delecio es a duplikacio merete, valamint transzlokaciok eseten az erintett partner kromoszomak egyuttesen hatarozzak meg.Subtelomeric regions of chromosomes are rich in genes; their rearrangements cannot be identified by traditional chromosome analysis. Since these subtelomeric aberrations are responsible for about 7% of cases with mental retardation, their detection is important both from the diagnostic point of view and to prevent recurrence in the family. Subtelomeric chromosomal alterations can be detected by fluorescence in situ hybridization. Based on international criteria, 35 out of 59 patients with mental retardation have been selected. Subtelomeric rearrangements were revealed in 6 patients (5 familial cases, 1 new onset) whereas the subtelomeric FISH result was normal in 29 cases. Deletion of 8pter and duplication of 12pter were detected in 2 patients, while a deletion of 21qter and duplication of the 10pter due to an unbalanced translocation were found in 3 other cases. Finally, a new onset deletion of 3qter was observed in 1 patient. In order to clarify the origin of chromosome aberrations, 12 healthy family members were also examined, 5 of them carried balanced translocations. We concluded that the phenotype is mostly influenced by the size of regions involved in deletion/duplication and - in case of translocations - by the associated chromosomal abnormalities.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Subtelomeric 6.7 Mb Trisomy 10p and 5.6 Mb Monosomy 21q Detected by FISH and Array-CGH in Three Related Patients

Cryptic subtelomeric chromosomal aberrations are responsible for 5–10% of moderate/severe and 1% of mild intellectual disability. Unbalanced subtelomeric chromosomal rearrangements result in variable phenotypes which seem to be highly influenced by both the size of the duplication/deletion and the chromosomes involved in the translocation. We report on three related patients with moderate intellectual disability, language delay, hypotonia, facial dysmorphism, cardiac anomalies, scoliosis, and kyphosis in whom a familial (maternal) unbalanced submicroscopic translocation was found by subtelomeric fluorescence in situ hybridization (FISH). This rearrangement resulted in a partial trisomy 10pter and partial monosomy 21qter. The karyotype was 46,XY.ish der(21)t(10;21)(p14;q22.2). Confirmation of a 6.7 Mb size distal duplication of the p15.3–14 region of chromosome 10 and a 5.6 Mb distal deletion of the q22.2–22.3 region of chromosome 21 was obtained by array‐CGH. To our best knowledge, such a composition of subtelomeric unbalanced translocations has not yet been published. Detection of this aberration in successive pregnancies of carrier members of the family by prenatal FISH could prevent the recurrence of the disease. Furthermore, detection of the rearrangements and identification of genes located in the chromosomal regions involved might be of interest.

Helyi infiltrációs érzéstelenítés alkalmazása nagyízületi endoprotézisek beültetése során

INTRODUCTION Total hip and knee replacment surgeries are characterized by severe postoperative pain. Local infiltration analgesia is proved to be very effective. However this method has not been widely used in Hungary. AIM To evaluate the efficacy of the local infiltration analgesia with modified components in patients underwent total hip or knee replacement surgery. METHOD Data of 99 consecutive patients underwent primary total hip or knee replacement surgery were evaluated prospectively. In all the 99 surgeries modified local infiltration analgesia was applied. Postoperative pain reported on a visual analog scale was recorded as well as the need for further analgetics during the first 18 hours after surgery. The cost of the analgetic drugs was calculated. The control group comprised 97 consecutive patients underwent total hip or knee replacement, where local infiltration analgesia was not applied. Statistical analysis was done. RESULTS Patients received local infiltration analgesia reported significantly less pain (p<0.001). The need for postoperatively given analgetics was almost 50% less, and the cost of all postoperative analgetics was 47% less than in the control group. CONCLUSION In total hip and knee replacement surgeries the modified local infiltration analgesia decreases postoperative pain effectively and contribute to the early mobilization of the patients. Orv. Hetil., 2017, 158(9), 352-357.Absztrakt: Bevezetes: A nagyizuleti endoprotezis-beultetesek jelentős posztoperativ fajdalommal jarnak. A lokalis infiltracios analgesia hatekony, de koltseges eljaras, hazankban nem terjedt el szeles korben. Celkitűzes: Modositott osszetetelű helyi infiltracios fajdalomcsillapitassal szerzett tapasztalataink bemutatasa. Modszer: Primer csipő-, illetve terdizuleti totalis endoprotezis-beultetesen atesett 99 paciens adatait dolgoztuk fel prospektive. Valamennyi paciens a műtet soran helyi infiltracios fajdalomcsillapitasban reszesult, amelyhez az ajanlasokhoz kepest modositott osszetetelű koktelt alkalmaztunk. A posztoperativ első 18 oraban orankent rogzitettuk a betegek altal a fajdalomskalan bejelolt fajdalomintenzitast. Feljegyeztuk, hogy a betegek milyen analgetikus terapiaban reszesultek meg posztoperative. Koltseghatekonysagi szamitast is vegeztunk. Kontrollcsoportkent 97 teljes primer csipő-, illetve terdprotetizalt paciens szolgalt. Eredmenyek: Az infiltracios erzestelenitesben reszesult betegek fa...

Torticollis in 15q11.2 Microdeletion Syndrome: a Rare Association in Angelman-like Syndromes

15q11-13 chromosome region contains five breakpoints (BP1-BP5). Chromosomal rearrangements are common in this region. The microdeletion of BP1-BP2 region represents the 15q11.2 microdeletion syndrome associating with variable phenotype. We investigated a ten years old boy with hypotony. His motoric functions, speech and intellectual development were delayed. He suffered from epilepsy and showed dysmorphic features. Some of these dysmorphic features such us epicanthus and the clynodactyly of the fifth fingers can be observed in Angelman or Prader-Willi syndromes but have not been described in the 15q11.2 microdeletion syndrome so far. He has congenital torticollis that has been described earlier neither in this microdeletion syndrome nor in Prader-Willi - Angelman syndromes. Our aim is to find the possible mechanisms leading to the phenotype using Metilation Specific - Multi Ligand Probe Assay, Polimerase Chain Reaction and Array Comparative Genomic Hybridization. The 15q11.2 microdeletion syndrome represents an example for the incomplete penetrance and variable expressivity. Further genetic changes, such as other defective genes, further copy number variations, variability in non-coding regions, the mRNA quantity, environmental effects and epigenetic modification may also influence on the severity of the symptoms. We suggest to classify the symptoms into two groups (major and minor criteria). Depending on the existing minor criteria, this syndrome could be identified as Angelman-like or Prader-Willi-like syndromes.

Altered lipid subfraction profile and impaired antioxidant defense of high-density lipoprotein in Smith-Lemli-Opitz syndrome

Background:Smith-Lemli-Opitz syndrome (SLOS) is a rare disease caused by biallelic mutation in the 7-dehydrocholesterol (7DHC) reductase gene. High oxidizability of 7DHC and the appearance of small-sized low-density lipoprotein (LDL) subfractions indicate increased endogenous oxidative stress that is counterbalanced by natural antioxidant defense mechanisms including the high-density lipoprotein (HDL)-associated paraoxonase-1 (PON1) enzyme. PON1 prevents lipoproteins from oxidative modifications; however, PON1 activity and the distribution of lipoprotein subfractions have not been studied in SLOS.Methods:7DHC levels and PON1 arylesterase activities were measured spectrophotometrically in 11 SLOS patients and 10 healthy children. Lipoprotein subfractions were detected by polyacrylamide gel electrophoresis.Results:Compared to controls, there was a shift towards the small-dense LDL subfraction and the large HDL subfraction in SLOS. PON1 arylesterase activity was significantly decreased in SLOS patients and correlated negatively with the proportion of small-dense LDL subfraction and the proportion of large HDL subfraction. Significant positive correlations were detected between PON1 arylesterase activity and the ratios of intermediate and small HDL subfractions.Conclusions:Decreased PON1 activity and the deleterious shift in the distribution of lipoprotein subfractions may contribute to the impaired antioxidant status observed in SLOS. Monitoring of serum PON1 arylesterase activity may be a complementary biomarker in SLOS.

A Koleszterin-Bioszintezis Veleszuletett Zavara: A Smith-Lemli-Opitz-Szindroma

Smith-Lemli-Opitz syndrome is an autosomal recessive mental retardation and multiple malformation syndrome caused by deficiency of the 7-dehydrocholesterol reductase, the enzyme catalyzing the last step in cholesterol biosynthesis. The authors summarize the pathophysiology, epidemiology, clinical picture, diagnostics and therapy of the disease based on a review of the international literature. Since 2004, fourteen patients have been diagnosed with Smith-Lemli-Opitz syndrome in Hungary, which suggests an underdiagnosis of the disease based upon estimated incidence data. Due to deficiency of the 7-dehydrocholesterol reductase, serum cholesterol concentration is low and 7-dehydrocholesterol concentration is elevated in blood and tissues; the latter being highly specific for the syndrome. Detection of disease-causing mutations makes the prenatal diagnosis possible. The clinical spectrum is wide, the most common symptom is syndactyly of the second and third toes. Standard therapy is cholesterol supplementation. Recent publications suggest that oxidative compounds of 7-dehydrocholesterol may play a role in the pathophysiology of the disease as well.Smith-Lemli-Opitz syndrome is an autosomal recessive mental retardation and multiple malformation syndrome caused by deficiency of the 7-dehydrocholesterol reductase, the enzyme catalyzing the last step in cholesterol biosynthesis. The authors summarize the pathophysiology, epidemiology, clinical picture, diagnostics and therapy of the disease based on a review of the international literature. Since 2004, fourteen patients have been diagnosed with Smith-Lemli-Opitz syndrome in Hungary, which suggests an underdiagnosis of the disease based upon estimated incidence data. Due to deficiency of the 7-dehydrocholesterol reductase, serum cholesterol concentration is low and 7-dehydrocholesterol concentration is elevated in blood and tissues; the latter being highly specific for the syndrome. Detection of disease-causing mutations makes the prenatal diagnosis possible. The clinical spectrum is wide, the most common symptom is syndactyly of the second and third toes. Standard therapy is cholesterol supplementation. Recent publications suggest that oxidative compounds of 7-dehydrocholesterol may play a role in the pathophysiology of the disease as well.

[Results of clinical and genetic diagnosis of rare diseases in the Eastern region of Hungary (2007-2013)].

INTRODUCTION 80% of rare diseases have a genetic origin, and 50% manifest themselves as congenital anomalies. Their adequate health care includes early recognition of genetic anomalies and prevention of recurrence. AIM The aims of the authors were to provide correct diagnoses to patients with multiple congenital anomalies with or without mental retardation attending to the outpatient clinic of the Clinical Genetics Center at the University of Debrecen in the time interval between August 1, 2007 and March 31, 2013, establish the possibility of prenatal diagnosis, assess the distribution of different genetic mechanisms in the background of rare genetic diseases, compare them with international data, and develop an algorithm for the diagnostic approach of rare genetic diseases applicable in Hungary. METHOD Clinical data and genetic results of patients were evaluated, and patients were categorized into one of the ten proposed etiological groups, based on which the distribution of genetic causes was defined. RESULTS Clinical diagnosis was achieved in 64.3% of patients, confirmed genetic diagnosis in 37.8%, while 35.7% of patients remained undiagnosed. Several dysmorphic syndromes and metabolic disorders were first diagnosed in Hungary, two of which unique in the literature. CONCLUSIONS In the centre of the authors the diagnostic effectiveness of chromosome aberrations exceeds the international standards, that of known microdeletions and dysmorphic syndromes meets international data, and the genetic diagnosis of mendelian disorders and submicroscopic copy number changes remain below international figures.