GENETIC CAUSES OF INFERTILITY

SAŽETAK Posljednjih godina došlo je do naglog razvoja dijagnostičkih metoda kojima se može otkriti genetski uzrok neplodnosti, a time procijeniti i rizik prijenosa genetskog poremećaja. Razvojem tehnika potpomognute oplodnje moguće je liječiti veoma teške oblike neplodnosti koji nose značajno veći rizik prijenosa određenih molekularnih promjena na potomstvo. Kod takvih slučajeva veoma je važno otkriti mutacijsku promjenu, jer se time pruža mogućnost davanja precizne genetske informacije o rizicima nasljeđivanja i načina prenatalnog određivanja genotipa ploda. S obzirom na to da ne postoje jedinstvene smjernice u pristupu dijagnostike i liječenja neplodnosti, nastoje se izdvojiti skupine visokorizičnih neplodnih bolesnika prema njihovim kliničkim osobitostima koje će biti podvrgnute rutinskim dijagnostičkim postupcima u svrhu otkrivanja uzroka neplodnosti. Za određene genske poremećaje, kao što su cistična fibroza i sindrom fragilnog X, poznata je uloga u etiopatogenezi neplodnosti. Najnovija molekularna istraživanja genskih mutacija, posebice u β-podjedinici FSH i LH gena, pokazuju svoju dijagnostičku i kliničku vrijednost kod otkrivanja genetskih uzroka neplodnosti.ABSTRACT During the last few years, there is a rapidly expansion of new diagnostic techniques improving the detection of genetic causes of infertility. It is followed by a precise risk assessment of genetic change transmission and possibility to chose the most adequate methode of medically assisted reproduction. The treatment of severe forms of both male and female infertility became possible by the development of very sofisticated in vitro fertilising techniques. Such patients carry the significantly higher risk of having mutational change in one of the disease genes involved in regulation of human reproduction. The detection of mutation in these patients improve the treatment and prognostic assessment in potential future pregnancies. The unique guidelines for the diagnosis and the treatment of infertile couples still doesn’t exist. There are several categories of patients selected by clinical features and associated with some forms of genetic abnormalities. For particular genetic disorders such as cystic fibrosis and fragile X syndrome, their role in pathogenesis of infertility is very well known. Every day, new genetic mutations associated with infertility are discovered. The recent studies involving investigations of significance of molecular mutations, particulary in the gene for the β-subunit of FSH and LH , showed thier diagnostic and clinical value in evaluation both male and female infertility

Advantages and limitations of invasive prenatal diagnosis

Prenatalna dijagnostika kromosomskih poremećaja podrazumijeva citogenetičku analizu stanica ploda dobivenih jednom od rutinskih invazivnih tehnika - biopsijom korionskih resica, placentocentezom, amniocentezom ili kordocentezeom. Izbor tipa invazivne dijagnostike ovisi ponajprije o individualnoj procjeni čimbenika rizika za rađanje kromosomski bolesnog djeteta, a zatim i o riziku lošeg ishoda, odnosno gubitka trudnoće kao najteže komplikacije invazivnog zahvata. Problemi koji se mogu javiti u prenatalnoj dijagnostici kromosomskih poremećaja su pojava kromosomskog mozaicizma te razlikovanje pravog od pseudomozaicizma, rizik neuočavanja suptilnih kromosomskih razmještanja zbog ograničenja razlučivanja svjetlosnog mikroskopa, nemogućnost detekcije kriptičkih mozaičnih oblika te zagađenje majčinim stanicama. U tim se slučajevima uz klasičnu citogenetičku analizu provodi i neka od metoda molekularne dijagnostike. Poseban izazov u prenatalnoj dijagnostici su blizanačke trudnoće. Uvođenjem metoda potpomognute oplodnje i novih protokola stimulacije, značajno se povećao broj višeplodnih trudnoća. Razlikovanje monozigotnih od dizigotnih blizanaca kod dikorijalnih diamnijskih blizanaca istog spola može se precizno odrediti usporednom analizom mikrosatelitnih lokusa. Mehanizmima kao što je postzigotno nerazdvajanje može doći do nastajanja različitih kromosomskih statusa, čak i kod monozigotnih blizanaca. Zato je amniocenteza tehnika izbora, a uzorci se trebaju uzeti iz svake amnijske vreće, posebice ako jedan ili oba blizanca imaju ultrazvučno dijagnosticirane abnormalnosti.Prenatal diagnosis of chromosomal abnormalities comprises cytogenetic analysis of fetal cells obtained after one of the routine invasive techniques, i.e. chorionic villus sampling, placentocentesis, amniocentesis, or cordocentesis. The choice of the type of invasive procedure is primarily based on individual combined calculation of all risk factors of having a chromosomally abnormal child. An important factor for the patient to decide to undergo an invasive procedure is the chance of pregnancy loss as the worst scenario. The problems that can arise in prenatal diagnosis of chromosomal disorders are diff erent types of mosaic forms and diffi culty to distinguish true fetal mosaicism from pseudomosaicism, the risk of missing a subtle anomaly due to the limitation of resolution of light microscopy, and maternal cell contamination. In these cases, some of molecular diagnostic procedures are performed. There is an increasing number of twin pregnancies after stimulation protocols and use of some of the techniques of in vitro fertilization procedures in the infertility treatment. Diff erentiation of monoyzgotic and dizygotic pregnancies is possible by applying microsatellite analysis. Chromosomal discordance in twin pregnancies in most cases is caused by postzygotic nondisjunction, and is recorded both in monozygotic and dizygotic twins. Amniocentesis is the technique of choice, and karyotyping from both amniotic sacs is recommended, especially in cases where one fetus has major structural malformations

Prenatal Diagnosis of 18p Deletion and Isochromosome 18q Mosaicism in a Fetus with a Cystic Hygroma

Although, deletion of short arm of chromosome 18 is one of the most frequent autosomal terminal deletions, mosaic form of 18p deletion is infrequently observed. Furthermore, prenatally detected cases of 18p deletion and isochromosome 18q mosaicism are extremely rare. Herein, we present a case of del(18p)/i(18q) mosaicism, prenatally detected after chorionic villus sampling. A 37-year-old woman was referred for prenatal diagnosis because of fetal septated cystic hygroma measuring 4.3 mm. Cytogenetic analysis showed a mosaic 46,XX,del(18)(p11.2)/46,XX,i(18)(q10) karyotype in both, short- and long-term culture. Parents elected to terminate the pregnancy. Fetal mosaic karyotype was confirmed by chromosomal analysis of cultured skin fibroblasts. Molecular characterization of chromosome 18 structural aberrations was performed by fluorescence in situ hybridization (FISH). Considering variable ultrasound findings among cases with del(18p)/i(18q) mosaicism, we emphasized that first and second trimester ultrasound screening examinations for fetal malformations, followed by cytogenetic and molecular evaluations, are very important in the management of prenatally detected cases

Genomic diagnostic algorithms in families with neurodevelopmental disorders

Genetska testiranja kod pacijenata s neurorazvojnim poremećajima vrlo su važna za postavljanje konačne dijagnoze. Prema trenutnim smjernicama prve metode izbora uključuju komparativnu genomsku hibridizaciju na mikropostroju te genetsko testiranje fragilnog X sindroma. Cjeloeksomsko sekvenciranje koje omogućuje detekciju jednonukleotidnih varijanti u kodirajućim regijama gena predstavlja sljedeći dijagnostički korak. Dijagnostički prinos komparativne genomske hibridizacije na mikropostroju raste do 15%, a cjeloeksomskog sekvenciranja čak do 40%. Međutim, kod velikog broja pacijenata (čak do 50%) etiologija neurorazvojnih poremećaja ostaje nerazjašnjena. Danas su poznati mnogi genetski mehanizmi koje nije moguće ustanoviti rutinskim dijagnostičkim metodama, a uključuju varijante broja kopija u nekodirajućim regulacijskim DNA regijama, varijante broja kopija koje utječu na strukturu i funkciju topoloških domena, duboke intronske varijante u kodirajućim genima kao i metilacijske obrasce u genomu. Navedene genske promjene moguće je detektirati novijim tehnologijama kao što je cjelogenomsko sekvenciranje i mapiranje topoloških domena (Hi-C sekvenciranje). Cjelogenomskim sekvenciranjem moguće je odrediti i precizne točke loma u naoko balansiranim kromosomskim razmještanjima. Uvođenje cjelogenomskog sekvenciranja u rutinsku dijagnostiku genetskih bolesti svakako bi povećalo dijagnostički prinos i omogućilo postavljanje dijagnoze za velik broj pacijenata s neurorazvojnim poremećajima. S napredovanjem tehnologije i sve većom dostupnošću cjelogenomskog sekvenciranja, u skoroj budućnosti se očekuje i povećanje genomskih baza koje bi omogućile interpretaciju velike količine podataka koju ova metoda generira, čime bi se ubrzalo uvođenje ove moćne metode u rutinsku medicinsku skrb kod pacijenata s genetskim poremećajima.Genetic testing in patients with neurodevelopmental disorders is very important for making a final diagnosis. According to the current guidelines, the first line methods include Microarray-based Comparative Genomic Hybridization and genetic testing of Fragile X Syndrome. Whole-exome sequencing, which allows detection of single-nucleotide variants in the coding regions of genes, represents the next diagnostic step. The diagnostic yield of Comparative Genomic Hybridization is up to 15%, while for whole-exome sequencing it increases up to 40%. However, in a large number of patients (up to 50%), the etiology of neurodevelopmental disorders remains unexplained. Even though there are many familiar genetic mechanisms, some of them cannot be established by routine diagnostic methods, such as copy number variants in non-coding regulatory DNA regions, copy number variants that affect structure and function of topologically associating domains (TADs), deep intronic variants in coding genes as well as genome-wide methylation patterns. The aforementioned genetic changes can be detected with newer technologies such as whole-genome sequencing and mapping of TADs (Hi-C sequencing). With whole-genome sequencing it is possible to determine precise breakpoints in apparently balanced chromosomal arrangements. Therefore introduction of whole-genome sequencing in the routine diagnostics of genetic diseases would certainly increase the diagnostic yield and enable setting up diagnosis for a large number of patients with neurodevelopmental disorders. With the advancement of technology and the increasing availability of whole-genome sequencing, a rise in the amount of available data in genomic databases is expected in the near future. This would allow the interpretation of the large amount of data generated by this method and accelerate the introduction of this powerful method into routine medical care for patients with genetic disorders

Association of Angiotensin-Converting Enzyme Insertion-Deletion Polymorphism with Preeclampsia

The aim of this study was to determine if insertion-deletion polymorphism of angiotensin-converting enzyme is a risk factor for the development of preeclampsia. Sixty women with preeclampsia and 50 normotensive pregnant women were included in this study. Preeclampsia was defined as blood pressure > 140/90 mmHg in a previously normotensive women with proteinuria >300 mg/L in a 24-hours. Twelve women also had preeclampsia in previous pregnancy. The genotyping of polymorphism in the intron 16 of the angiotensin-converting enzyme was performed by the polymerase chain reaction followed by the agarose electrophoresis. The patients were divided into three groups according to the presence (I) or absence (D) of insertional polymorphism (II, ID, and DD). Genotype distribution and allele frequencies were compared by Mantel-Haenszel c 2 testing. The frequency of DD genotype was not significantly higher in women with preeclampsia (26/60)than in the control group (14/50, p=0.096). The D allele frequency was significantly higher in 17 women with preeclampsias who required delivery before 34 weeks of pregnancy (0.735), than in 43 women in whom obstetric complications took place after 34 weeks of pregnancy (0.56, p=0.036). The D allele frequency was 0.83 in women having recurrent preeclampsia, i.e. significantly higher compared with women, who were for the first time, experienced preeclampsia (0.57, p=0.013). This study showed a significantly positive association between D allele frequency and risk of recurrent preeclampsia and preterm delivery before 34 weeks of pregnancy. The deletion genotype could be an important contributing factor for an early onset and recurrent preeclampsia

De Novo Case of a Partial Trisomy 4p and a Partial Monosomy 8p

The extent of clinical expression in cases of segmental aneuploidy often varies depending on the size of the chromosomal region involved. Here we present clinical and cytogenetic findings in a 5-month old boy with a duplication of a chromosomal segment 4p16.1→4pter and a deletion of a chromosomal segment 8p23.1→8pter. His karyotype was determined by applying classical GTG banding and FISH method (WHCR region, centromere 4, centromere 8, telomere 8p) as 46,XY,der(8)t(4;8)(p16.1;p23.1).ish der(8)t(4;8)(D8S504-,WHCR+,D8Z2+)dn. Parents are not related and have normal karyotypes, indicating de novo origin. We have compared similarity of the clinical features in our proband to other patients carrying only a duplication of the distal part of 4p or a deletion of distal part of 8p or similar combination described in the literature

Parasomnias: differential diagnostic aproach and importance of polysomnography

Parasomnija je poremećaj spavanja s visokom prevalencijom u općoj populaciji. Bez obzira na to što je parasomnija većinom benigne naravi, najčešće predstavlja vrlo neugodan i nepoželjan fenomen u spavanju u djece i odraslih, snižava kvalitetu života, a može biti krivo protumačena i liječena kao epilepsija. Diferencijalna dijagnostika je posebice izazovna u osoba s napadajima s predominantnim kompleksnim motoričkim ponašanjem koji nastaju tijekom hipermotorne epilepsije s noćnim napadajima, kada može dovesti do nepotrebnih i skupih pretraga te neuspješnog liječenja. Koegzistencija parasomnije i epilepsije u istoj obitelji i/ili u iste osobe upućuje na zajedničke neurofiziološke temelje. U ovom preglednom radu opisane su kliničke i neurofiziološke osobitosti najčešćih parasomnija, genetske osnove te pouzdani elementi u diferencijalnoj dijagnostici parasomnija i noćnih epileptičkih napadaja. Prikazana je dijagnostička vrijednost anamnestičkih podataka, video EEG polisomnografije i dijagnostičkih skala. U djece s epilepsijom i parasomnijama, pogotovo u djece koja imaju noćne napadaje, ne smije se zanemariti postojanje dnevne pospanosti i poteškoća spavanja. Svaku osobu s epilepsijom i s dnevnom pospanošću ili sumnjom i na neepileptogene noćne događaje treba poslati na cjelonoćnu polisomnografiju. Liječenje komorbiditeta koji pogoršavanju spavanje u osoba s epilepsijom i kronofarmakologija epilepsije osiguravaju bolji uspjeh liječenja epilepsije. Pravovremeno prepoznavanje postojanja parasomnije omogućava pravilnu procjenu njezinoga zdravstvenog značenja za osobu i utjecaja na kvalitetu života. Uvođenje medicine spavanja u kurikulume diplomskog i poslijediplomskog obrazovanja omogućava adekvatno obrazovanje zdravstvenih radnika i stjecanje potrebnih vještina za rad u specijaliziranim laboratorijima i odjelima za poremećaje spavanja.Parasomnias are sleep disorders with a high prevalence in the general population. Regardless of the fact that parasomnia is mostly benign in nature, it often represents a very unpleasant and undesirable phenomena during sleep in children and adults, lowering the quality of life, and can be misinterpreted and treated as epilepsy. Differential diagnosis is especially challenging in people with seizures with dominant complex motor behavior that occur during hypermotor epilepsy with nocturnal seizures when it can lead to unnecessary and expensive examinations and unsuccessful treatment. The coexistence of parasomnia and epilepsy in the same family and/or in the same person points to the common neurophysiological foundation. This review paper describes the clinical and neurophysiological features of the most common parasomnias, their genetic basis, and reliable elements in the differential diagnosis of parasomnias and nocturnal epileptic seizures. The diagnostic value of anamnestic data, video EEG polysomnography and diagnostic scales is presented. In children with epilepsy and parasomnias, especially in children who have nocturnal seizures, daytime sleepiness and difficulty sleeping should not be ignored. Any person with epilepsy and with daytime sleepiness or suspicion of non-epileptogenic nocturnal events should be sent for overnight polysomnography. Treatment of comorbidities that worsen sleep in people with epilepsy and chronopharmacology of epilepsy ensure better success in the treatment of epilepsy. Timely recognition of the existence of parasomnia allows a proper assessment of its health significance and its impact on the quality of life. The introduction of sleep medicine into the curricula of the graduate and postgraduate education enables adequate education of health workers and the acquisition of the necessary skills for working in specialized laboratories and units for sleep disorders

ZNAČAJ INTRAKARDIJALNIH EHOGENIH ŽARIŠTA FETALNOG SRCA: SADAŠNJE SHVAĆANJE I KLINIČKA VRIJEDNOST

Objective. To estimate the degree of risk of the echogenic intracardiac foci (IEF) for fetal chromosomopathies and to determine its association with structural anomalies of the fetus. Material and methods. During the period of two years 190 pregnant patients had been send for fetal echocardiography. Examination had been performed by transvaginal¬ (12–17 weeks of gestation) or transabdominal approach (18 weeks or more of gestation). Results. IEF was observed in 17 fetuses, multifocal appearance was found in 2 out of 17 fetuses. In 3 cases IEF had resolved during the 8 weeks period of time. Additional structural anomalies were detected in 11 fetuses. In 2 fetuses trisomy 21 had been confirmed. Conclusion. A single soft marker as IEF is commonly encountered during the second trimester among the fetuses with chromosomal aberation. As do many sonographic markers IEF can be resolved during the pregnancy and often can be found in normal fetuses.Cilj rada je bio na vlastitom uzorku utvrditi u kojoj mjeri ultrazvučni nalaz hiperehogenih intrakardijalnih žarišta (IEF) pridonosi dijagnostici kromosomopatija i strukturalnih anomalija. Uzorak i metode. Tijekom dvije godine 190 trudnica između 12. i 39. tjedna trudnoće je primljeno radi fetalne ehokardiografije. Pregled je obavljen vaginalnom sondom od 5 MHz pri trudnoćama 12.–17. tjedna ili zavinutom abdominalnom sondom od 3,5 MHz nakon 17. tjedna trudnoće. Rezultati. IEF su nađeni u 17 fetusa, multifokalni u 2 od njih. U 3 fetusa su IEF u roku od osam tjedana nestali. U 11 fetusa su nađene dodatne strukturalne anomalije. Trisomija 21 je potvrđena u 2 fetusa. Zaključak. IEF su »meki« ultrazvučni biljezi fetalne aneuploidije, često su prolazni, a nalaze se i u eukariotičnih fetusa