Variable fragment length allele-specific polymerase chain reaction (VFLASP), a method for simple and reliable genotyping.

Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific position in the genome. Until now, 585 million SNPs have been identified in the human genome, and therefore, a widely applicable method is desirable to detect a specific SNP. Herein we report a simple and reliable genotyping assay, which seems to be suitable for medium and small size laboratories, as well, to easily genotype most of the SNPs. In our study, all of the possible base variations (A-T, A-G, A-C, T-G, T-C, G-C) were tested to prove the general feasibility of our technique. The basis of the assay is a fluorescent PCR, in which both allele-specific primers, differing only at the 3' end according to the sequence of the SNP, were present, and the length of one of them was modified with 3 bp by adding an adapter sequence to the 5' end of that primer. The competitive presence of both allele-specific primers excludes the false amplification of the absent allele (which can happen in simple allele-specific PCR (AS-PCR)) and ensures the amplification of the proper allele(s). Unlike other complicated genotyping methods that use of manipulation of fluorescent dyes for genotyping, we apply an approach based on the length of amplicons from different alleles to differentiate between them. In our experiment (named variable fragment length allele-specific polymerase chain reaction (VFLASP)), the investigated six SNPs, containing the six available base variations, gave clear and reliable results after detecting the amplicons by capillary electrophoresis.

Intrauterine diagnosis and pathology of fetal choroid plexus carcinoma--a case study.

This is a case report of a 39-year-old pregnant woman whose fetus was found to have a large hydrocephalus on routine prenatal ultrasound at the 29th gestational week. A 56 mm × 73 mm mass was detected in the fetal brain arising from the brainstem and invading the third cerebral ventricle. On the subsequent fetal cranial MRI, T2-weighted image the tumor measured 55 mm × 50 mm × 48 mm and had a non-homogeneous consistency and irregular contours. Elective cesarean section was performed during gestational week 32, delivering a male fetus with a cranial circumference of 46 cm (normal circumference, 30 cm) and a birth weight of 2920 g. The infant expired 4h following delivery. Autopsy revealed a carcinoma of the choroid plexus. Our case, like others, suggests that MRI is more accurate than prenatal ultrasound in prenatal brain tumor diagnosis. More precise morphological detail is provided by MRI, which improves surgical planning and survival.

[Prenatal diagnosis of central nervous system malformations]. / A központi idegrendszer fejlodési rendellenességeinek praenatalis diagnosztikája.

The prenatal diagnosis of fetal malformations have been the subject of numerous publications in the literature. This has dramatically increased in the last 15 years, mainly due to the advent of high-resolution ultrasound. In addition adequate guidelines issued by professional organizations have encouraged the universal approach to the imaging of fetal anatomy as well as malformations. One of the most significant groups of the fetal anomalies is the central nervous system malformation. Due to its prevalence and severity the praenatal diagnostics of central nervous system malformations got basic significance. In this review we attempted to summarize the recent informations concerning the prenatal diagnostics of the central nervous system anomalies.

Isolated and ventriculomegaly-associated cases of spina bifida in genetic counseling: focus on fetal pathology.

Cases of spina bifida alone and in association with ventriculomegaly represent important but different malformations according to clinical characteristics. In our study, we analyzed the data on pregancies terminated because of isolated cases (n=307) and ventriculomegaly-associated cases (n=372) of spina bifida. In spina bifida cases in association with hydrocephalus, positive obstetric history was found approximately 1.5 times more frequently than in the isolated ones. The incidence of positive genetic history was nearly two-fold in the latter cases. In isolated cases of spina bifida, associated malformations were more common than in cases of spina bifida and ventriculomegaly together. The most frequent associated malformations were those of the urogenital system (in cases of spina bifida: 11.1%; in cases of SB+V: 9.14%). The risk of recurrence of SB+V is significantly higher than that of isolated SB (8.9% vs. 2.1%). It can be concluded that positive genetic history is more common in cases of isolated spina bifida. Malformations out of the nervous system are more commonly observed in cases of isolated spina bifida. During the prenatal diagnostics of spina bifida, sonography must focus on malformations of the urogenital system.

Spontaneous abortion in multiple pregnancy: focus on fetal pathology.

Multiple pregnancy with its wide array of medical consequences poses an important condition during pregnancy. We performed perinatal autopsy in 49 cases of spontaneous abortion resulting from multiple pregnancies during the study period. Twenty-seven of the 44 twin pregnancies ending in miscarriage were conceived naturally, whereas 17 were conceived through assisted reproductive techniques. Each of the 5 triplet pregnancies ending in miscarriage was conceived through assisted reproductive techniques. There was a positive history of miscarriage in 22.4% of the cases. Monochorial placentation occurred more commonly in multiple pregnancies terminating with miscarriage than in multiple pregnancies without miscarriage. A fetal congenital malformation was found in 8 cases. Three of these cases were conceived through assisted reproductive techniques, and 5 were conceived naturally. Miscarriage was due to intrauterine infection in 36% of the cases. Our study confirms that spontaneous abortion is more common in multiple than in singleton pregnancies. Monochorial placentation predicted a higher fetal morbidity and mortality. In pregnancies where all fetuses were of male gender, miscarriage was more common than in pregnancies where all fetuses were female. Assisted reproductive techniques do not predispose to the development of fetal malformations.

Comparison of prevalence of toxoplasma and cytomegalovirus infection in cases with fetal ultrasound markers in the second trimester of pregnancy.

OBJECTIVES: To evaluate the prevalence of toxoplasma and cytomegalovirus (CMV) infections in cases of ultrasound anomalies detected in the second trimester of pregnancy. METHODS: Serological examinations for toxoplasma and CMV infection were carried out in 655 cases with sonographic findings suggestive of fetal infection, 612 cases with single ultrasound markers and 43 cases with two or more markers. RESULTS: In cases of single ultrasound markers, serological examination diagnosed recent toxoplasma infection in 107/612 cases (17.5%) and recent CMV infection in 75 cases (12.3%). Recent toxoplasma infection accounted for 13.8% (52/377) of the intracranial sonographic findings and 23.9% (45/188) of the abdominal findings, whereas recent CMV infections accounted for 12.2 (46/377) and 11.7% (22/188), respectively. Recent CMV infection with sonographic manifestations had higher rates of intracranial than intra-abdominal sonographic findings (46/75 or 61% vs 22/75 or 29%), whereas recent toxoplasma infection with sonographic manifestations had similar rates of intracranial (52/107 or 49%) and intra-abdominal (45/107 or 42%) findings. In cases of two or more ultrasound markers, serological examination diagnosed recent toxoplasma infection in 12/43 cases (27.9%) and recent CMV infection in 10/43 cases (23.3%). CONCLUSIONS: Ultrasound findings suspicious for toxoplasma and CMV infection are not pathognomonic for either pathologic entity.

Positive predictive value of prenatal ultrasonography confirmed by post-mortem investigations.

OBJECTIVE: To analyse the positive predictive value of ultrasonography based on the results of post-mortem investigations in respect of the most common malformations. STUDY DESIGN: We processed the details of 683 cases affecting the nervous, cardiovascular, urinary and skeletal systems, and the abdominal/thoracic walls. RESULTS: In four of the investigated groups of malformations, the proportion of sonographic diagnoses completely coinciding with the post mortem results was found to be approximately 70%, but it was found to be much lower (32%) in urinary malformations. The cumulative proportion of coinciding sonographic and post mortem diagnoses was 63%, while completely incorrect ultrasonographic diagnoses amounted to 17%. CONCLUSIONS: Sonography is an essential method for the diagnosis of the majority of fetal malformations, but post-mortem examinations yield an indispensable quality control as well as additional information to ultrasound examinations.

Non-syndromic malformations of the central nervous system in twin pregnancies: diagnostic and other clinical features of importance.

OBJECTIVE: The incidence of central nervous system malformations is higher among twins. Our aim was to summarize information about these malformations in twin pregnancies. STUDY DESIGN: Based on a sample originating from the biggest genetic centre in Hungary between January 1990 and December 2008, we examined the data of 42 twin pregnancies associated with non-syndromic malformations of the central nervous system. RESULTS: The involvement of monozygotic fetuses and dizygotic ones of the same gender was found to be 62.5%. Usually only one of the fetuses was affected (57.1%), while the other one was healthy. The male-to-female ratio was 0.75. Identical and fraternal twins were found in 68.4% and 31.6% of the cases, respectively. In the pregnancies of our study the malformation was diagnosed before the 24th gestational week in 90% of the cases. Polyhydramnios (54.8%) was the most commonly associated non-central nervous system malformation. CONCLUSION: Our findings suggest that, in addition to placentation and gestational age, the position of the affected fetus with relation to the uterine orifice is of great importance in determining whether selective abortion is an option in deciding about the outcome of pregnancies affected by craniospinal malformation.

Etiology, prenatal diagnostics and outcome of ventriculomegaly in 230 cases.

OBJECTIVE: The aim of this study was to review and summarize the information regarding the etiology, diagnostics and outcome of ventriculomegaly. METHODS: The study included 230 cases of ventriculomegaly examined between 1979 and 2000. The main diagnostic criterion for ventriculomegaly was the transverse diameter of the ventricular atrium at the level of the glomus of the chorioid plexus measuring >10 mm, irrespective of gestational age. RESULTS: Gender distribution (male:female ratio: 0.98) coincided with that of the general population. In 32% of the cases (72/230), the history was positive; 6% (12/230) had a positive genetic history, while 26% (60/230) were associated with pathological obstetric events. The incidence rate of ventriculomegaly in the patients' history was found to be 2.61% (6/230). In nearly 60% of the cases included in this study, ventriculomegaly was diagnosed before the 24th week of pregnancy. Fresh fetal infection confirmed by Toxoplasma PCR real-time examination was diagnosed only in cases of severe ventriculomegaly. Based on the measurement of the diameter of the atrium of the lateral ventricle, severe and mild ventriculomegaly was diagnosed in 142/230 (61.7%) and 88/230 cases (38.3%), respectively. Termination of pregnancy was significantly more frequent in cases of severe than of mild ventriculomegaly (92 vs. 66%). CONCLUSIONS: The importance of positive obstetric and/or genetic history should be emphasized as it is in direct relationship with the increased incidence of this malformation. Regarding the practice of ultrasonography, mild ventriculomegaly (transverse diameter of the lateral ventricle <15 mm) has a much better prognosis than the severe form (transverse diameter of the lateral ventricle >15 mm) of the malformation. Based on the ultrasonographic diagnosis of ventriculomegaly, TORCH serological examination is also recommended since treating toxoplasmosis by medication may have a promising prognosis for the pregnancy. In cases of isolated ventriculomegaly alone, intrauterine karyotyping is not necessarily indicated, but in cases where ventriculomegaly is associated with other genetic disorders karyotyping should definitely be performed. Since ventriculomegaly is not incompatible with postnatal life by itself, the decision about the fate of the pregnancy is largely dependent on the presence of other organic disorders.

[Common malformations in fetopathologic investigations between 1995 and 2006. Accuracy of ultrasonography confirmed by post mortem investigations]. / Gyakoribb fejlodési rendellenességek a fetopatológiai vizsgálatok anyagában 1995-2006 között. Az ultrahang-diagnosztika hatékonysága a post mortem vizsgálatok tükrében.

UNLABELLED: Fetopathologic investigations are of great importance since they are aimed at assessing the risks of a malformation to recur in a mother's future pregnancy. AIMS: The authors wished to assess and analyse the accuracy of ultrasonography based on the results of fetopathologic investigations in view of malformations of higher prevalence. STUDY DESIGN: The authors have processed the details of 683 cases affecting the nervous, cardiovascular, urinary and skeletal systems, and the abdominal/thoracic walls detected at our department in the period of 1995-2006. RESULTS: No significant differences could be justified as far as the major statistical parameters of maternal and gestational ages at the time of the diagnosis were concerned. There were one or another positive detail in the history in a quarter of malformations affecting the central nervous, cardiovascular and skeletal systems and in one sixth of the cases with disorders of the urinary tract as well as the abdominal/thoracic wall. Urinary tract and cardiovascular malformations were far more common among male fetuses, while moderate female dominance could be observed in malformations of the central nervous system and of the abdominal/thoracic wall. In the four investigated groups of malformations, the proportion of sonographic diagnoses completely coinciding with the post mortem results was found to be approximately or more than 70%, but it was found to be much lower (38%) in urinary malformations. Based on the full sample, the cumulative proportion of coinciding sonographic and fetopathologic diagnoses were more than 63%, while completely incorrect ultrasonographic diagnoses amounted to 18%. CONCLUSIONS: (Even multiple) sonographic investigations are to be performed in a genetic centre if urinary tract malformation with subsequent oligohydramnion is detected. Since the associability of omphalocele and chromosome aberrations has been an established fact, and because some of the cases with omphalocele have been diagnosed as gastroschisis, it may be advisable to perform chromosome investigations in ultrasonographically diagnosed cases of gastroschisis. In cases of VSD, echocardiography should be performed in high-risk pregnancies even if ultrasonography cannot reveal any changes in the patients.

Major diagnostic and pathological features of iniencephaly based on twenty-four cases.

Iniencephaly is quite a rare malformation the etiology of which is still not fully understood. In the majority of cases it is a grave and lethal condition. It is often complicated by other abnormalities affecting the central nervous system (spina bifida, anencephaly), but malformations involving other organs and systems may also be observed. Based on 24 cases the authors have surveyed the diagnostics of iniencephaly with special regard to the disorders affecting the central and non-central nervous systems. In addition, they have compared the results of prenatal diagnostics and pathological investigations. In the sample, maternal age ranged between 17 and 42 (median 24) years. Positive obstetrical-gynecological and genetic findings in the patients' history have been reported in 4 and 2 cases, respectively. In these cases, the maternal serum alpha-fetoprotein (AFP) values ranged between 0.7 and 3.9 (median 2.0) MoM, while the amniotic fluid AFP values were between 0.9 and 2.7 (median 1.4) MoM. Spina bifida (50%) and anencephaly (42%) were the most commonly occurring complications affecting the central nervous system. Among the non-central nervous system disorders, malformations of the abdominal (omphalocele) and thoracic walls (diaphragmatic hernia) were found most frequently and the tendency to develop associated polyhydramnios was also very high (75%). Pathological investigations revealed developmental disorders such as cleft lip and palate, ventricular septal defect and facial dysmorphism, which are difficult to detect using ultrasonography.

Risk of chromosome abnormalities in the presence of bilateral or unilateral choroid plexus cysts.

OBJECTIVES: To evaluate the rate of chromosome abnormalities in cases of uni- and bilateral choroid plexus cysts (CPCs). METHODS: A total of 10,875 ultrasound (US) examinations were performed in the second trimester, and 435 cases with CPC (4%) were found. After genetic counseling, 45 patients decided not to undergo karyotyping. The authors performed a chromosome analysis in 390 cases of CPCs. RESULTS: The total risk of chromosome abnormalities was 3.59% (n = 14) and risk of trisomies was 2.05% (n = 8). Trisomy 18 was found in 6 cases (1.54%), trisomy 21 in 1 case (0.26%), and trisomy 9 in 1 case (0.26%). The risk of 45,X karyotype was 0.77% (n = 3). One case of 47,XXY karyotype and 2 cases with other chromosome abnormalities were found. In 212 unilateral cases there were 7 with chromosome abnormalities (3.3%). In 178 bilateral cases there were 7 with abnormal karyotypes (3.93%). The CPC was associated with additional fetal US anomalies (with or without polyhydramnios/oligohydramnios) in 112 cases; chromosome abnormalities were detected in 4 cases (3.57%). 66 cases were associated with polyhydramnios/oligohydramnios but not with other fetal US anomalies; 3 cases of abnormal karyotypes were found (4.55%). The CPC was isolated in 212 cases and 7 cases were associated with chromosome disorders (3.3%). CONCLUSIONS: US plays an important role in prenatal diagnostics. Further genetic counseling is recommended in cases with CPCs.

[Craniospinal malformations in a twelve-year fetopathological study; the efficiency of ultrasonography in view of fetopathological investigations]. / Craniospinalis malformatiók 12 év foetopathologiai vizsgálatainak anyagában; az ultrahang-diagnosztika hatékonysága a foetopathologiai vizsgálatok tükrében.

BACKGROUND: Craniospinal malformations represent a heterogeneous group of congenital malformations by their morphology and etiology alike. Certain craniospinal malformations could be diagnosed as early as the dawn of ultrasonography and this group of malformations has been the focus of attention ever since. AIMS: The aim of the authors was to review the main characteristics of craniospinal malformations, as well as to evaluate the efficiency of ultrasonography based on autopsy examinations during twelve years. STUDY DESIGN: The current study comprises the details of 339 pregnancies terminated by induced abortion for craniospinal malformation between 1995 and 2006. RESULTS: Maternal median age was 27 +/- 5.8 years, ranging from 15 to 47 years. In 24.5% of the cases, there was a positive obstetrical-gynaecological or genetic history. In 68.1% of the cases, ultrasonographic and autopsy findings were completely identical; in 24.2% a partial coincidence was found, but autopsy allowed for further diagnoses, while in 26 cases (7.7%) different findings were obtained by prenatal ultrasonography and fetopathological investigations. In half of the latter 26 cases, induced abortion was suggested due to hydrocephalus confirmed by ultrasonography but not justified by autopsy or the autopsy revealed the presence of other craniospinal malformation(s). CONCLUSION: It can be concluded that--in view of the diagnostic efficiency of fetopathological investigations--the ultrasonographic diagnosis of hydrocephalus should be interpreted independently from other craniospinal malformations on the basis of principles of ultrasonographic methodology and at different times.

Craniospinal malformations in a twelve-year fetopathological study; the efficiency of ultrasonography in view of fetopathological investigations.

BACKGROUND: Craniospinal malformations represent a heterogeneous group of congenital malformations by their morphology and etiology alike. Certain craniospinal malformations could be diagnosed as early as the dawn of ultrasonography and this group of malformations has been the focus of attention ever since. AIMS: : The aim of the authors was to review the main characteristics of craniospinal malformations, as well as to evaluate the efficiency of ultrasonography based on autopsy examinations during twelve years. STUDY DESIGN: The current study comprises the details of 339 pregnancies terminated by induced abortion for craniospinal malformation between 1995 and 2006. RESULTS: Maternal median age was 27+/-5.8 years, ranging from 15 to 47 years. In 24.5% of the cases, there was a positive obsterical-gynecological or genetic history. In 68.1% of the cases, ultrasonographic and autopsy findings were completely identical; in 24.2% a partial coincidence was found, but autopsy allowed for further diagnoses, while in 26 cases (7.7%) different findings were obtained by prenatal ultrasonography and fetopathological investigations. In half of the latter 26 cases, induced abortion was suggested due to hydrocephalus confirmed by ultrasonography but not justified by autopsy or the autopsy revealed the presence of other craniospinal malformation(s). CONCLUSION: It can be concluded that - in view of the diagnostic efficiency of fetopathological investigations - the ultrasonographic diagnosis of hydrocephalus should be interpreted independently from other craniospinal malformations on the basis of principles of ultrasonographic methodology and at different times.

Chlamydia prevalence and correlates among female adolescents in Hungary.

PURPOSE: To evaluate the prevalence and the behavioral and historical determinants of genital chlamydial infection among adolescent females in Hungary. METHODS: A total of 214 consecutive, unselected, self-referred, sexually active, nonpregnant female individuals aged 16-20 years were evaluated by polymerase chain reaction through the use of questionnaires. RESULTS: The prevalence of chlamydial infection within this population was 7.9%. We find that the most important correlates of chlamydial infection were at least three lifetime sexual partners (p < .005), two or more sexual partners in the preceding 3 months (p < .05), and symptoms of vaginitis (p = .002). CONCLUSIONS: The high prevalence of chlamydia in this study population may justify universal testing in Hungary.

Risk of recurrence in major central nervous system malformations between 1976 and 2005.

OBJECTIVE: The goal of the current publication is to review isolated central nervous system malformations (CSMs) using a database in excess of 75 000 cases, with special regard to the risk of recurrence of these malformations alone or in combination. METHODS: In the period between 1 January 1976 and 31 December 2005, among the 75 320 documented cases, consultations were requested due to earlier isolated CSMs in the patients' histories in 3030 cases (4.2%). Processing the data we only considered disorders of genetic origin, and that was why we excluded the cases due to intrauterine infection. Monogenically inherited malformations were also excluded from the analysis. The diagnosis of the malformations was based on the prenatal diagnosis of ultrasonography as well as the findings of the foetopathological examination. RESULTS: In 65% of the cases, the couples sought counselling because of malformation in a previous pregnancy. In these cases, the risk of recurrence was thought to be 5.2%, while in the case of two affected children this figure stood at 21.9%. Analysing the values for the risk of recurrence in 5-year periods, neural tube defects (NTDs) (particularly anencephaly and spina bifida) showed a detectable decrease, which could be attributed to a growing use of folic acid supplementation around the time of conception and during pregnancy. CONCLUSION: There is a clear decrease of risk of recurrence of NTDs, while in the case of the other CSMs in this study, there is no noteworthy chronological change in their risk of recurrence.

Neural tube defects in the sample of genetic counselling.

OBJECTIVE: This study was conducted to evaluate the major demographic details, diagnostical and clinical features, as well as the risk of recurrence of cases with the major types of neural tube defects (NTD). We also examined the efficiency of ultrasonography based on autopsy examinations during 26 years. METHODS: The investigations were made into the sample of 743 NTD diagnosed between 1 January 1976 and 31 December 2002. A computerized database was used to sum up the available information about the individual cases; in addition to surveying the couples' major demographic details, we also had the opportunity to collect detailed information about the history, diagnostics (ultrasound) and outcome of the pregnancies as well as the results of the autopsies during the investigation. RESULTS: In the 743 cases of NTD, maternal and paternal median ages turned out to be 23.7 years (+/-5.22 years) and 28.7 years (+/-5.81 years), respectively. The male:female ratio was 0.78. Comparable samples of anencephaly and spina bifida allowed for the conclusion that a positive genetic history was equally often found while a positive obstetrical history was almost twice as common in anencephaly. The sensitivity of the maternal serum-alpha fetoprotein (AFP) screening test is the highest in anencephaly and lowest in encephalocele. While the majority of cases of anencephaly were diagnosed before the 24th gestational week, examples of diagnosing spina bifida and encephalocele at a later time could also be found. Among the associated malformations other than those of the central nervous system special mentioning should be made of fetal pyelectasia, cleft palate as well as diaphragmatic herniation. No pathological karyotypes were found in association with encephalocele or spina bifida, but anencephaly was accompanied with trisomy 21 and trisomy 18 in one case each. Anencephaly was found to have the highest risk of recurrence in both nervous system malformations and malformations other than those of the nervous system. Sonography proved to be the most reliable method in cases of enecephalocele. CONCLUSION: The respective median values of maternal and paternal age show that aetas has no role in the occurrence of NTDs. NTDs are more common among girls. Positive genetic, obstetrical and medical findings are of great importance in the incidence of NTDs. Although reliable to only a limited extent, maternal serum-AFP tests are considered to be useful and necessary in screening NTDs, while sonography is the gold standard method in recognizing these frequent malformations. The knowledge of the eventual associated malformations is mainly important in certain cases of spina bifida, which may also yield a good post-natal prognosis. Our data obtained from the sample of 26 years also confirm that the periconceptional administration of folic acid reduces the incidence and risk of recurrence of NTDs.