Herediter neuromusculáris betegségek szűrése molekuláris genetikai módszerekkel hazai roma populációban = Screening of hereditary neuromuscular disorders in the Roma population living in Hungary

Recent medical genetic research has identified a number of novel, or previously known, but rare conditions, caused by private founder mutations. The Finnish and Ashkenazi Jew populations provide the best examples for identifying genes in unique genetic disorders. In these populations, research efforts and high-level medical services resulted in intense improvements of medical care and in organization of population-based screening programs. Hereditary disorders of the Roma populations are known for a long time. The genetic background of these diseases has been established by extensive molecular genetic studies. The Romas represent 6% of the Hungarian population and live under extremely bad health conditions. Therefore, our aim was to map the incidence of the hereditary neuromuscular disorders among the Hungarian Roma population. Moreover, we intended to provide proper information, genetic counseling and possible prevention strategies for the families at risk, which should represent a primer task in public health. Because of our experience in neuromuscular disorders, we choose six, frequent, autosomal recessive disorders for these clinical and genetic studies: hereditary motor and sensory neuropathy type Lom (HMSNL), hereditary motor and sensory neuropathy type Russe (HMSNR), congenital cataracts facial dysmorphism syndrome (CCFDN), limb-girdle muscular dystrophy 2C (LGMD2C), congenital myasthenic syndrome (CMS) and spinal muscular atrophy (SMA). Following identification of the founder mutations, the possibility of prenatal diagnosis and carrier screening for family members will contribute to the decrease of the recurrence risk for these severe, mostly untreatable disorders

A de novo atypical ring sSMC(22) characterized by array CGH in a boy with cat-eye syndrome.

BACKGROUND: Microduplications 22q11 have been characterized as a genomic duplication syndrome mediated by nonallelic homologous recombination between region-specific low-copy repeats. Here we report on a 19 years old boy with intellectual disability having an unexpected structurally complex ring small supernumerary marker chromosome (sSMC) originated from a larger trisomy and a smaller tetrasomy of proximal 22q11 harboring additional copies of cat eye syndrome critical regions genes. RESULTS: PRINCIPAL CLINICAL FEATURES WERE: anorectal and urogenital malformations, total anomalous pulmonary venous return with secundum ASD, hearing defect, preauricular pits, seizure and eczema. The proband also presented some rare or so far not reported clinical findings such as hyperinsulinaemia, severe immunodeficiency and grave cognitive deficits. Chromosome analysis revealed a mosaic karyotype with the presence of a small ring-like marker in 60% of cells. Array CGH detected approximately an 1,2 Mb single and a 0,2 Mb double copy gain of the proximal long arm of chromosome 22. The 1,3 Mb intervening region of chromosome 22 from centromere to the breakpoints showed no copy alteration. The karyotype of the patient was defined as 47,XY,+mar[60]/46,XY[40].ish idic r(22)(q11.1.q11.21) x 4.arr 22q11(17,435, 645-18,656,678) x 3,(17,598,642-17,799,783) x 4 dn. CONCLUSIONS: The present report is the first one with a detailed description of clinical presentation in a patient carrying an atypical size ring sSMC (22) analyzed by array CGH. The specialty of the finding is emphasized by the fact that although the patient had a mosaic sSMC and the amplified region was smaller than in typical cat eye syndrome cases, the clinical presentation was severe

Molecular cytogenetic analysis of Xq critical regions in premature ovarian failure.

BACKGROUND: One of the frequent reasons for unsuccessful conception is premature ovarian failure/primary ovarian insufficiency (POF/POI) that is defined as the loss of functional follicles below the age of 40 years. Among the genetic causes the most common one involves the X chromosome, as in Turner syndrome, partial X deletion and X-autosome translocations. Here we report a case of a 27-year-old female patient referred to genetic counselling because of premature ovarian failure. The aim of this case study to perform molecular genetic and cytogenetic analyses in order to identify the exact genetic background of the pathogenic phenotype. RESULTS: For premature ovarian failure disease diagnostics we performed the Fragile mental retardation 1 gene analysis using Southern blot technique and Repeat Primed PCR in order to identify the relationship between the Fragile mental retardation 1 gene premutation status and the premature ovarion failure disease. At this early onset, the premature ovarian failure affected patient we detected one normal allele of Fragile mental retardation 1 gene and we couldn't verify the methylated allele, therefore we performed the cytogenetic analyses using G-banding and fluorescent in situ hybridization methods and a high resolution molecular cytogenetic method, the array comparative genomic hybridization technique. For this patient applying the G-banding, we identified a large deletion on the X chromosome at the critical region (ChrX q21.31-q28) which is associated with the premature ovarian failure phenotype. In order to detect the exact breakpoints, we used a special cytogenetic array ISCA plus CGH array and we verified a 67.355 Mb size loss at the critical region which include total 795 genes. CONCLUSIONS: We conclude for this case study that the karyotyping is definitely helpful in the evaluation of premature ovarian failure patients, to identify the non submicroscopic chromosomal rearrangement, and using the array CGH technique we can contribute to the most efficient detection and mapping of exact deletion breakpoints of the deleted Xq region

Izomdystrophiák differenciál-diagnosztikai vizsgálata molekuláris genetikai, valamint immunhisztokémiai és immunoblot analízisek segítségével = Differential diagnostic study of muscle dystrophies by molecular genetic, immunohystochemical and Western-blot analyses

A DMD/BMD betegségben célul tűzték ki a cDNS próbák alkalmazását a hordozósági státusz vizsgálatára és a deléciók pontos méretének meghatározására. Az újonnan bevezetett MLPA módszer segítségével a teljes dystrophin gén feltérképezésére, bizonyos pont mutációk kiszűrésére, a deléciók és duplikációk pontos analízisére, továbbá a hordozóság gyors szűrésére is vállalkozhattak. Összesen 120 beteg és 89 női hozzátartozó molekuláris analízisét végezték el a pályázat támogatásával. Az FSHD betegségben a komplex genetikai háttér miatt szükséges a molekuláris diagnosztikus kritériumok finomítása. A betegség hátterében álló epigenetikus változások kulcsfontosságúak lehetnek fenotípus kialakulásában. A betegség pathomechanizmusában a D4Z4 szekvenciáktól proximálisan elhelyezkedő gének megváltozott transzkripciójának van szerepe, amelynek vizsgálatára metilációs analízist alkalmaztak. A klinikai diagnózis megerősítésére összesen 90 beteg és 46 tünetmentes hozzátartozó DNS mintáját analizálták. Az LGMD betegségcsoport a betegek differenciál-diagnosztikája jelenleg csak specifikus fehérjedetektálási módszerekkel lehetségesek. Ezeket a módszereket alkalmazták azokban az esetekben, ahol az FSHD és DMD/BMD molekuláris genetikai vizsgálata negatív volt. A vizsgálati periódusban 122 beteg izombiopsziás mintája érkezett, ebből 56 esetben immunhisztokémiai, míg 20 esetben kiegészítő western blot analízis történt. További, DNS szintű analízisek 14 esetben azonosították a patogén mutációt. | The first aim of the study was to introduce cDNA probes for the analysis of DMD/BMD in order to detect carrier status and exact deletion borders. Additionally, the new MLPA technique was first introduced by us in Hungary and enabled the detection of the entire dystrophin gene with highlights on specific pointmutations, exact analysis of the deletions and duplications and moreover, on efficient screening of the carrier status. During the project, 120 affected persons and 89 female relatives were screened. In case of the FSHD molecular genetic criteria have to be improved because of the complexity of the disorder. Epigenetic influences may play a crutial role in modulating phenotype. Altered transcriptional activities of neighbouring genes proximal to the D4Z4 sequences have a key place in the pathomechanism, therefore, this effect was studied by methylation analysis. For confirmation of the clinical diagnosis 90 patients and 46 asymptomatic family members were genetically nalysed. The differential diagnosis of the heterogenous group of LGMD requires at first the specific analysis of the dystrophin-associated glycoproteins. This was used in the case of patients where the establishment of the genetic diagnosis of DMD/BMD or FSHD failed. Muscle biopsies and blood samples of 122 patients were sent to the laboratory; in total 56 immunohistochemical and 20 Western blot analyses have been performed. In 14 patients the exact pathogenic mutation has been identified by DNA sequencing

A microarray-komparatív genomhibridizálás (arrayCGH) praenatalis alkalmazása. Javaslat a hazai bevezetésre = Chromosomal microarray comparative genome hybridization (arrayCGH) in prenatal settings. Proposal for Hungarian application in clinical practice

Absztrakt: Az invazív mintavétel kapcsán elvégzett hagyományos magzati kromoszómavizsgálat a mai napig a praenatalis diagnosztika alapvető vizsgálómódszere. Felhasználásának a fénymikroszkópos vizsgálat felbontási képessége szab határt. A kariotipizálással nem felismerhető, szubmikroszkópos kromoszóma-rendellenességek, microdeletiók és microduplicatiók, kópiaszám-variációk (CNV-k) vizsgálatára a nagy felbontású molekuláris vizsgálóeljárások biztosítanak lehetőséget. A kromoszomális összehasonlító microarray-vizsgálat (array-komparatív genomhibridizálás – arrayCGH) alkalmas az anyai életkortól függetlenül előforduló kópiaszám-variációk prae- és postnatalis kimutatására. A módszer a fejlett országok orvosi gyakorlatában rutinszerűen alkalmazott eljárás a magzati diagnosztikában. Az elmúlt egy évtized külföldi eredményei alapján alkalmazása ultrahangeltérést nem mutató magzatok esetén körülbelül 1–2%, strukturális ultrahangeltérést mutató magzatoknál körülbelül 5–7% többlet genetikai információval szolgál a hagyományos kromoszómavizsgálattal szemben. Közleményünkben áttekintjük az arrayCGH módszerét, praenatalis alkalmazásának nemzetközi gyakorlatát, s javaslatokat és indikációs kört fogalmazunk meg a módszer praenatalis használatának magyarországi bevezetésére. Orv Hetil. 2019; 160(13): 484–493. | Abstract: Invasive prenatal testing and conventional G-banding chromosome analysis have been considered to be the gold standard of fetal cytogenetic diagnosis. Standard karyotyping is, however, constrained by the limits of the resolution of using a microscope. The advantage of molecular karyotyping, array based methods is the evaluation of sub-microscopic copy number changes across the whole genome in a single analysis. The application of array comparative genome hybridization has greatly increased the detection of pathogenic chromosomal abnormalities in prenatal settings. Based on available data in the international literature of the last decade, the clinical utility of arrayCGH is the recognition of some 1–2% and 5–7% additional genetical information compared to metaphase karyotype alone in fetuses without ultrasound anomaly and in fetuses with ultrasonographically detected malformations, respectively. Thus arrayCGH improves the prenatal diagnosis of genetic abnormalities mainly in fetuses with structural sonographic findings. In the present paper we review the literature of chromosomal microarray and make a proposal for the application of the method in Hungarian prenatal genetical practice. Orv Hetil. 2019; 160(13): 484–493

A ritka kromoszóma-rendellenességek és a fetoplacentaris mozaikosság jelentősége a praenatalis diagnosztikában a nem invazív szűrővizsgálatok tükrében

Introduction and objective: To determine the prevalence of microscopically visible de novo atypical chromosomal aberrations and fetoplacental mosaicism in a prenatal tertial referral center, and to investigate the maternal and fetal characteristics in connection with genomewide non-invasive prenatal screening. Method: Retrospective cohort study from 2014 to 2019 of pregnancies with invasive genetic analysis. Results: In the cohort of 2504 cases, the proportion of CVS was 53.3%. We diagnosed 200 chromosomal aberrations (8%), including 13.5% of de novo rare chromosomal aberrations (14 rare autosomal trisomies, 6 polyploidies, 5 structural aberrations and 2 small supernumerary marker chromosomes). The rate of fetoplacental mosaicism was 12.4%/77.8% in common/atypical chromosomal aberrations (p = 0.001) and confined to placenta in 17/40 cases. Associated ultrasound abnormalities were abnormal nuchal translucency and major malformations in 58% and 35% with common trisomies and 11% (p = 0.006) and 67% (p = 0.047) with true mosaic cases of rare abnormalities, respectively. Major ultrasound malformation was facial dysmorphism with rare aberrations. Potential application of genomewide non-invasive prenatal test in atypical chromosomal aberrations presumably would have been false-positive in 12 cases (44%), false-negative in 1 case (3.7%), and would have early detected 2 cases of rare autosomal trisomies (7.4%) without ultrasound anomalies. Conclusion: Structural ultrasound malformations with normal nuchal translucency thickness may be indicative of rare chromosomal aberrations. Application of genomewide non-invasive prenatal test is an alternative of early diagnostic methods with a potential of discordant results due to mosaicism. Knowledge about the presence of fetoplacental mosaicism influences risk estimation and genetic counseling, detailed cytogenetic characterization is of utmost importance. © Szerző(k)

Complex X chromosome rearrangement associated with multiorgan autoimmunity

BACKGROUND: Turner syndrome, a congenital condition that affects 1/2,500 births, results from absence or structural alteration of the second sex chromosome. Turner syndrome is usually associated with short stature, gonadal dysgenesis and variable dysmorphic features. The classical 45,X karyotype accounts approximately for half of all patients, the remainder exhibit mosaicism or structural abnormalities of the X chromosome. However, complex intra-X chromosomal rearrangements involving more than three breakpoints are extremely rare. RESULTS: We present a unique case of a novel complex X chromosome rearrangement in a young female patient presenting successively a wide range of autoimmune diseases including insulin dependent diabetes mellitus, Hashimoto's thyroiditis, celiac disease, anaemia perniciosa, possible inner ear disease and severe hair loss. For the genetic evaluation, conventional cytogenetic analysis and FISH with different X specific probes were initially performed. The complexity of these results and the variety of autoimmune problems of the patient prompted us to identify the exact composition and breakpoints of the rearranged X as well as methylation status of the X chromosomes. The high resolution array-CGH (assembly GRCh37/hg19) detected single copy for the whole chromosome X short arm. Two different sized segments of Xq arm were present in three copies: one large size of 80,3 Mb from Xq11.1 to Xq27.3 region and another smaller (11,1 Mb) from Xq27.3 to Xq28 region. An 1,6 Mb Xq27.3 region of the long arm was present in two copies. Southern blot analysis identified a skewed X inactivation with approximately 70:30 % ratios of methylated/unmethylated fragments. The G-band and FISH patterns of the rearranged X suggested the aspect of a restructured i(Xq) chromosome which was shattered and fortuitously repaired. The X-STR genotype analysis of the family detected that the patient inherited intact maternal X chromosome and a rearranged paternal X chromosome. The multiple Xq breakages and fusions as well as inverted duplication would have been expected to cause a severe Turner phenotype. However, the patient lacks many of the classic somatic features of Turner syndrome, instead she presented multiorgan autoimmune diseases. CONCLUSIONS: The clinical data of the presented patient suggest that fragmentation of the i(Xq) chromosome elevates the risk of autoimmune diseases

Clinical and genetic findings in Hungarian pediatric patients carrying chromosome 16p copy number variants and a review of the literature

The short arm of chromosome 16 (16p) is enriched for segmental duplications, making it susceptible to recurrent, reciprocal rearrangements implicated in the etiology of several phenotypes, including intellectual disability, speech disorders, developmental coordination disorder, autism spectrum disorders, attention deficit hyperactivity disorders, obesity and congenital skeletal disorders. In our clinical study 73 patients were analyzed by chromosomal microarray, and results were confirmed by fluorescence in situ hybridization or polymerase chain reaction. All patients underwent detailed clinical evaluation, with special emphasis on behavioral symptoms. 16p rearrangements were identified in 10 individuals. We found six pathogenic deletions and duplications of the recurrent regions within 16p11.2: one patient had a deletion of the distal 16p11.2 region associated with obesity, while four individuals had duplications, and one patient a deletion of the proximal 16p11.2 region. The other four patients carried 16p variations as second-site genomic alterations, acting as possible modifying genetic factors. We present the phenotypic and genotypic results of our patients and discuss our findings in relation to the available literature.info:eu-repo/semantics/publishedVersio

Hordozóságszűrés Duchenne-/Becker- izomdystrophiával érintett családokban [Carrier detection in families affected by Duchenne/Becker muscular dystrophy]

Duchenne/Becker muscular dystrophy is a severe, recessive, X-linked neuromuscular disease with an incidence of 1/3500 (Duchenne type) and 1/30,000 (Becker type) in newborn boys. The gene responsible for the Duchenne/Becker muscular dystrophy phenotype is located at Xp21 and its 427 kD) protein product is called dystrophin. Deletions, point mutations and rarely duplications can occur almost anywhere in the DMD gene, which makes the molecular diagnosis difficult. Multiple polimerase chain reactions detect 95% of deletions in affected males [2, 4], but are not suitable for carrier detection in female relatives. Southern-blot analysis with six different cDNA probes covers the whole 14 kb dystrophin transcript and allows the detection of female carriers by comparing the intensity of the signals corresponding to the different exons. This method is time consuming compared to the newly introduced multiple ligation-dependent probe amplification method. Multiple ligation-dependent probe amplification is a method suitable for relative quantification of several DNA sequences in one reaction. The authors report results on 93 cases where the carrier status was analysed simultaneously by cDNA hybridisation and multiple ligation-dependent probe amplification technique. In 42 cases the carrier state was confirmed and in this carrier population the authors additionally detected two cases with duplication, two cases with one copy of the whole dystrophin gene and three manifest carrier females. On the basis of these results the MLPA technique, which has been newly introduced in Hungary, proved to be a sensitive and quick method for the detection of carrier state in the DMD/BMD disease. Moreover, the exact deletion or duplication border can be detected and as a result, prediction on the phenotype can be given. This will provide the right therapeutic intervention for the affected patients in the future

Dystrophim gene Analysis in Hungarian Duchenne/Becker muscular dystrophy families - Detection of carrier status in symptomatic and asymptomatic female relatives

A comprehensive study of the Hungarian Duchenne/Becker muscular dystrophy (DMD/BMD) families is presented. Deletions in the hot spots regions were identified by multiplex PCR, whereas rare Mutations were detected by Southern blot and multiplex ligation-dependent probe amplification (MLPA) techniques. DMD/BMD disease was confirmed and exact deletion borders were determined in 19 out of 135 affected males using multiplex PCR. Additional exons involved as well as rare exon deletions were identified by MLPA in 71 male patients, whereas duplications were observed in seven patients. In two DMD patients, the entire dystrophin gene and adjacent genes were deleted. Out of the 95 female relatives, 41 proved to be carriers, including three manifesting carrier females. Using MLPA method, a large portion of the Hungarian DMD/BMD patients and their female relatives were exactly genotyped. For the first time, the incidence and prevalence of asymptomatic and symptomatic female carriers in Hungary was estimated. (C) 2008 Elsevier B.V. All rights reserved