Triploid pregnancy-Clinical implications.

Triploidy is a life-limiting genetic aberration resulting from an extra haploid set of chromosomes of paternal (diandric triploidy) or maternal origin (digynic triploidy). Triploidy affects around 1%-2% of all conceptions. The majority of cases is miscarried at early developmental stages. In consequence of genomic imprinting, parental origin affects the phenotype of triploid pregnancies as well as the prevalence and spectrum of related maternal complications. Distinctive ultrasound features of both triploid phenotypes as well as characteristic patterns of biochemical markers may be useful in diagnosis. Molecular confirmation of the parental origin allows to predict the risk of complications, such as gestational trophoblastic neoplasia, hyperthyroidism, hypertension, or preeclampsia associated with the paternal origin of triploidy. Diagnosis of partial hydatidiform mole associated with diandric triploidy is challenging especially in the first trimester pregnancy loss due to the limitations of both histopathology and ultrasound. We present important clinical aspects of triploid pregnancies and indicate unresolved issues demanding further studies.

Distribution of diandric and digynic triploidy depending on gestational age.

PURPOSE: To establish the distribution of diandric and digynic triploidy depending on gestational age. METHODS: 107 triploid samples tested prospectively in a single genetic department during a four-year period were analyzed for parental origin of triploidy by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) (n=95) with the use of matching parental samples or by MS-MLPA (n=12), when parental samples were unavailable. Tested pregnancies were divided into three subgroups with regard to the gestational age at spontaneous pregnancy loss: <11 gestational weeks, 11-14 gestational weeks, and >14 gestational weeks. RESULTS: Diandric triploidy constituted overall 44.9% (46.5% in samples miscarried <11 gestational weeks, 64.3% in samples miscarried between 11 and 14 gestational weeks, and 27.8% in pregnancies which survived >14 gestational weeks). CONCLUSIONS: The distribution of diandric and digynic triploidy depends on gestational age. The majority of diandric triploid pregnancies is lost in the first trimester of pregnancy. In the second trimester, diandric cases are at least twice less frequent than digynic ones.

Usefulness of methylation-specific multiplex ligation-dependent probe amplification for identification of parental origin of triploidy.

Triploidy is a genetic aberration resulting from an extra haploid set of chromosomes of paternal (diandric) or maternal (digynic) origin. Diandric cases, opposite to digynic ones, may lead to gestational trophoblastic neoplasia (GTN) or generate maternal complications, therefore their identification is crucial, but reproducibility of traditionally used histopathological assessment is poor. The aim of the study was to analyse the usefulness of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) with probes for two differentially methylated regions (DMR) at chromosome 11p.15.5 for identification of the parental origin of triploidy. 84 triploid DNA samples were tested with MS-MLPA: 34 paternal cases (40.5%) and 50 maternal ones (59.5%) according to the reference results of QF-PCR. Methylation ratio (MR) was calculated. Reference values proposed by the MRC-Holland for diploid samples (MR 0.8-1.2) were used. The values outside these ranges were used to diagnose parental origin of triploidy-paternal (MR > 1.2) or maternal (MR < 0.8). The effectiveness of MS-MLPA was 94.0%. The mean MR in paternal triploidy was 1.7 (SD-0.25; n = 34) compared with 0.56 in maternal triploidy (SD-0.12; n = 50). MR values in paternal and maternal triploidy did not overlap. In five samples (6.0%) parental origin of triploidy could not be accurately established by MS-MLPA, probably due to the maternal cell contamination (MCC). MS-MLPA can be used as a convenient method for distinguishing between paternal and maternal triploidy without the necessity for parental samples testing. It enables adequate selection of the paternal triploid cases for follow up in order to exclude post-molar GTN.

Interaction between polymorphisms of the oxytocinergic system genes and emotion perception in inpatients with anorexia nervosa.

OBJECTIVE: The empirical literature describes the role of the oxytocinergic system in emotion perception (EP). Variants in the oxytocin (OXT) and oxytocin receptor genes have been associated with mental disorders, including anorexia nervosa (AN), that are characterized by difficulties in socioemotional functioning. Our study aimed to examine whether variability within the genes related to OXT pathways may play a role in facial EP in inpatients with AN. METHOD: Single nucleotide polymorphisms (SNPs) of the following genes: oxytocin receptor (rs2254298, rs53576), OXT (rs6133010), OXT-arginine-vasopressin (rs2740204), CD38 (rs6449197, rs3796863), and human leucyl/cystinylaminopeptidase (rs4869317) were genotyped in 60 AN female inpatients and 60 healthy control females (HCs). Associations between genetic polymorphisms and EP as well as clinical symptoms were examined. RESULTS: The AN group showed decreased EP abilities compared with HCs. SNPs of rs2740204, rs6133010, and rs53576 were associated with differences in EP in women with AN and in HCs. The SNP of rs4869317 was associated with the level of eating disorders symptoms in HCs. CONCLUSIONS: The OXT system may be involved in EP difficulties in AN. SNPs within genes related to OXT pathways may influence EP abilities. The leucyl/cystinylaminopeptidase rs4869317 SNP may be involved in the development of eating disorders psychopathology.

Deletions, not duplications or small mutations, are the predominante new mutations in the dystrophin gene.

Examination of the carrier state was performed in 744 unrelated mothers of the Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) probands with identified mutations in the dystrophin gene. Owing to that it was possible to assess frequency and type of new mutations in the gene. Contrary to the Japanese observations of Lee et al. published in this journal, we did not find significant differences in the carrier frequency between mothers of DMD and BMD patients. However, we found that new mutations in patients with deletions were significantly more frequent than in those with duplications and small mutations: of 564 unrelated patients with deletions, 236 (41.8%) carried new mutations, the respective values for duplications and small mutations were 21 of 95 patients (22.1%) and 18 of 85 patients (21.2%)-the differences highly significant (P<0.0001).

Prenatal diagnosis of Duchenne and Becker muscular dystrophies: Underestimated problem of the secondary prevention of monogenetic disorders.

AIM: The aim of this study was to analyze the influence of effective preconceptional testing for carrier status in women at risk for Duchenne and Becker muscular dystrophies (D/BMD) on the prenatal diagnosis. METHODS: A retrospective analysis of 201 prenatal tests was performed in 169 Polish women at risk, in regard to time of testing for carrier status (prior to conception or during pregnancy) and carrier status of tested women, including confirmed D/BMD carriers (n = 78; 46.2%), D/BMD non-carriers - tested for germline mosaicism risk (n = 23; 13.6%), and women at risk with uncertain carrier status (n = 68; 40.2%). RESULTS: Only 52.7% of women were tested for D/BMD carrier status prior to conception and in these women prenatal diagnosis was carried out more frequently in the first trimester of pregnancy (64.7% vs 47.8%; P = 0.035). The results of prenatal testing in male fetuses in pregnancies of confirmed D/BMD carriers and D/BMD non-carriers - tested for germline mosaicism risk were conclusive in all cases, whereas in women with uncertain carrier status, only 60.0% of results were conclusive. Eighty-five of 103 female fetuses (82.5%) were tested prenatally and in 31.8% of them fetal carrier status was confirmed. CONCLUSION: Carrier status testing in women prior to conception has a positive impact on the frequency of first-trimester prenatal diagnosis and known D/BMD carrier status on the effectiveness of prenatal diagnosis. Due to the low percentage of women tested effectively prior to conception, carrier status testing in the families at risk should be propagated (including possibility of prenatal diagnosis of female fetuses).

First trimester pregnancy loss: Clinical implications of genetic testing.

Around 10-15% of pregnancies result in a spontaneous first trimester miscarriage, which is most frequently caused by chromosomal abnormalities, mainly aneuploidies. Genetic analysis of pregnancy loss includes conventional G-banding karyotyping and various molecular methods. Apart from variable methodological limitations, the effectiveness of genetic analysis depends on the type and quality of the tested sample. To improve the reliability of genetic testing, we present methods of appropriate collection and pre-laboratory preparation of chorionic villi from first trimester miscarriage. We also discuss issues of maternal cell contamination, placental mosaicism and reciprocal and Robertsonian translocations in the context of interpretation of the results and genetic counseling.

Genetic polymorphisms and serum concentrations of adiponectin and resistin in anorexia nervosa and healthy controls - pilot study.

BACKGROUND: A possible role of adipokines in the regulation of body weight in patients with anorexia nervosa (AN) has been proposed. Polymorphisms in genes encoding adiponectin and resistin in AN have not been widely assessed, yet. OBJECTIVES: 1) Assessment the frequency of ADIPOQ c.45T>G, ADIPOQ c.276G>T polymorphisms in adiponectin and RETN c.62G>A, RETN c.-180C>G in resistin genes in AN patients and control group (C) 2) Analysis of correlation between these polymorphisms and serum ADP or RETN. METHODS: We examined 67 AN girls and 38 C aged 11-18. Analyses of polymorphisms in ADIPOQ and RETN genes were performed using RFLP method and adiponectin and resistin serum levels - with commercially available ELISA kits. RESULTS: In AN subjects, TT genotype in ADIPOQ c.276 polymorphism as well as GG genotype of RETN c.-180 were significantly more frequent than in CG. In ADIPOQ c.45 polymorphic site, TT alleles were the most frequent in both examined groups. In RETN c.62 GA and GG alleles distribution did not differ between the groups and the most frequently observed genotype was GG. The mean serum adiponectin level in AN was significantly higher and resistin - lower than in controls. There were no statistically significant relationships between serum adiponectin and resistin levels and allele frequency in polymorphisms ADIPOQ c.276 as well as RETN c.-180 in the examined groups. CONCLUSION: Differences in genotype frequencies of ADIPOQ c.276 and RETN c.-180 suggest a need for studies on a larger cohort of patients with AN.

Co-incidence of Turner syndrome and Duchenne muscular dystrophy - an important problem for the clinician.

INTRODUCTION: Turner syndrome is a relatively common chromosomal disorder which affects about one in 2000 live born females. Duchenne muscular dystrophy is an X-linked recessive disorder affecting 1:3600 live born males. Considering the above, the coexistence of these two diseases may occur only anecdotally. CASE PRESENTATION: Here, we report a 4 ½ year-old female with classical 45,X Turner syndrome who also had Duchenne muscular dystrophy caused by a point mutation in the dystrophin gene (c.9055delG). The patient showed the typical phenotype of Turner syndrome including distinctive dysmorphic features (short neck, low posterior hairline, wide position of nipples), aortic coarctation and feet lymphedema. Besides, she presented with an unusually early beginning of muscular dystrophy symptoms with infantile-onset motor developmental delay, intellectual disability and early calf muscular hypertrophy. CONCLUSION: The coexistence of an X-linked recessive disorder should be considered in women affected by Turner syndrome presenting with additional atypical clinical features.

The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations.

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).

MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy.

Duchenne/Becker muscular dystrophy (DMD/BMD) is a recessive, X-linked disorder caused by a mutation in the dystrophin gene. Deletions account for approximately 60-65% of mutations, duplications for 5-10%. The remaining cases are mainly point mutations. According to Monaco theory clinical form of the disease depends on maintaining or disrupting the reading frame. The purpose of the study was to determine frequency and location of deletions and duplications in the dystrophin gene, to determine the compliance between maintaining/disrupting the reading frame and clinical form of the disease and to check the effectiveness of MLPA (multiplex ligation-dependent probe amplification) in the detection of these mutations in hemizygous patients and heterozygous female carriers. The material is composed of combined results of molecular diagnosis carried out in years 2009-2012 in 180 unrelated patients referred with the diagnosis of DMD/BMD tested by use of MLPA. We identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45-54 and 3-21, whereas most duplications involved exons 3-18. The compliance with Monaco theory was 95% for deletions and 76% for duplications. Most of mutations in the dystrophin gene were localized in the hot spots - different for deletions and duplications. MLPA enabled their quick identification, exact localization and determination whether or not they maintained or disrupted the reading frame. MLPA was also effective in detection of deletions and duplications in female carriers.

[Non-invasive prenatal diagnosis of the most common aneuploidies with cell-free fetal DNA in maternal serum--preliminary results]. / Nieinwazyjna diagnostyka prenatalna najczestszych aneuploidii na podstawie plodowego DNA we krwi matki --doniesienie wstepne.

OBJECTIVES: The aim of the study was to present initial results of non-invasive prenatal diagnosis of common aneuploidies of chromosomes 21, 18 and 13 based on cell-free fetal DNA in maternal serum in high-risk patients, and to compare the results with routine karyotyping. MATERIAL AND METHODS: Before the invasive procedure, 10 ml of peripheral blood from 10 patients was collected to isolate cell-free fetal DNA and to perform a non-invasive fetal trisomy test (NIFTY provided by Beijing Genomics Institute, BGI, Shenzen, China). RESULTS: Three out of 10 samples showed an abnormal karyotype in traditional karyotyping. There were 9 conclusive NIFTY results. NIFTY detected 1 out of 2 trisomies 18. The quantity of cell-free fetal DNA in maternal plasma in the second probe with trisomy 18 was unsatisfactory fora conclusive NIFTY result. In 1 case traditional karyotyping revealed mosaicism impossible to detect with NIFTY

Two mutations in one dystrophin gene.

BACKGROUND AND PURPOSE: Duchenne/Becker muscular dystrophies (DMD/BMD) lead to progressive irreversible muscle deterioration caused by recessive mutations in the dystrophin encoding gene (Xp21.1). Approximately 60% of mutations are deletions, 10% are duplications and the remaining 30% are point mutations. The aim of the study is to present the rare occurrence of two pathogenic mutations (deletions or duplications) in one allele of the dystrophin gene. MATERIAL AND METHODS: DNA of patients from 1364 DMD/ BMD families was tested. Two techniques - PCR-multiplex and multiplex ligation-dependent probe amplification - were used to search for mutations in the dystrophin gene. RESULTS: Deletion was detected in 648 families and duplication was found in 74 families (analysis in progress). In two families, presence of two mutations in one gene was documented - in the first family two deletions were found (exons 45-49 and 60-61), and in the second family two duplications were detected (exons 2-7 and 50-59). One of the deletions disrupted the reading frame, and the other deletion retained the reading frame. Both duplications also retained the reading frame of the gene but in both families the disease took a severe course (DMD). In the family with two duplications prenatal diagnosis was also carried out, and carriership of both mutations was discovered in the female fetus. CONCLUSIONS: In the analyzed group of DMD/BMD families, the frequency of combined occurrence of two mutations in one gene was 2 per 722 (0.3%). The phenomenon of detected non-contiguous deletions and duplications is presented together with 31 similar cases published so far.

[Noninvasive prenatal diagnosis of trisomy 21, 18 and 13 using cell-free fetal DNA]. / Nieinwazyjna diagnostyka prenatalna trisomii 21,18 i 13 z wykorzystaniem wolnego pozakomórkowego DNA plodu.

Trisomy 21, 18 and 13 are the most common trisomies diagnosed in newborns. Screening methods consist of ultrasound and maternal serum markers. High risk for fetal aneuploidies is an indication for routine karyotyping, which requires collection of fetal tissue through amniocentesis or chorionic villous sampling. They are invasive procedures and carry a potential risk of miscarriage. The discovery of cell free fetal DNA (cffDNA) in maternal blood offered new opportunities for noninvasive prenatal diagnosis. The fraction of cell-free fetal DNA in total pool of cell-free DNA in maternal plasma is very low, therefore the analysis of cffDNA is very challenging. The introduction of massive parallel sequencing has enabled the application of noninvasive prenatal testing in the clinical practice and a variety of recent studies have proven its high efficacy in diagnosing common aneuploidies.

Multiplex ligation-dependent probe amplification (MLPA)--new possibilities of prenatal diagnosis.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a relatively new method of molecular diagnosis. It enables a relative quantitative assessment of up to 50 different PCR amplicons in one reaction by the use of a very small amount of examined DNA. Nowadays MLPA is becoming a very helpful tool in prenatal diagnosis and is widely used for the detection of aneuploidies, familial single gene disorders, common microdeletion syndromes, sub-telomeric alterations and identification of marker chromosomes in fetuses. This review demonstrates possible applications of MLPA in prenatal diagnosis.

Hereditary form of prion disease in Poland.

BACKGROUND AND PURPOSE: The aim of the study was to perform molecular analysis in a group of patients affected with prion disease. Diagnosis was based on results of clinical and/or histopathological examination of the brain. This is the largest investigation of this type performed so far in Poland. MATERIAL AND METHODS: Analysed material contained 36 cases of prion disease, including 35 cases of Creutzfeldt-Jakob disease and one case of Gerstmann-Sträussler-Scheinker disease, as well as two familial cases initially suspected of Huntington disease and Alzheimer disease. The control group consisted of 87 subjects. The most frequent known mutations in the PRNP gene were looked for, namely those in codons 102, 117, 178, 200, 217 and OPRI; the polymorphism Met/Val in codon 129 was also analysed. The methods applied were PCR-RFLP and DNA sequencing. RESULTS: The following mutations were found: E200K in 5 families, P102L in one family (previously identified), D178N in one family and 6OPRI in one family. Overall, mutations were detected in 17 persons (including 8 preclinical ones) from 8 pedigrees. Highly significant difference of codon 129 Met/Val heterozygosity frequencies was found between the affected subjects and the controls. Frequency of the familial form of prion disease in the material analysed was 14%. CONCLUSIONS: Screening for mutations in the PRNP gene should be performed in all diagnosed cases of prion disease and in cases of familial occurrence of early onset dementia of unknown aetiology. Families with identified mutations should be offered genetic counselling and informed of risks of blood and organs' donation.

Clinical Importance of aCGH in Genetic Counselling of Children with Psychomotor Retardation.

Introduction: The X and Y chromosomes are responsible for the determination and differentiation of the gonads, and their numerical and structural abnormalities may cause the abnormal development of secondary sex characteristics. The presence of abnormalities concerning X chromosome can also contribute to many genetically heterogeneous diseases associated with cognitive impairment and intellectual disability. Purpose: This study shows the effect of aberrations of the maternal X chromosome on the abnormal development of the child. Patients and Methods: Ten women aged 26 to 40 years were consulted in genetic counselling clinic and subsequently subjected to cytogenetic and molecular tests due to abnormal psychomotor development of their children, in whom structural aberrations of the X chromosome had been detected. Results: Two women were diagnosed with changes in karyotype: 46,X,der(X)t(X;Y)(p22.3;q11.2) in one and 46,X,inv(X)(p21.2q13). Five women were diagnosed with microduplications in the short arm of the X chromosome; dupXp22.31 in one, and in four women dupXp22.33. The remaining three women were diagnosed with duplication in the long arm of the X chromosome; dupXq25 in one and dupXq26.3 in two women. Conclusion: Genetic analysis of the X chromosome, based on cytogenetic and molecular methods of the highest available resolution, is extremely important in women with reproductive failure. These methods allow establishing accurately the breakpoints and rearrangements in chromosomes, and assessment of the copy number variation (CNV) can explain phenotypic variability with apparently similar aberrations. A more precise characterization of the alterations is necessary for the correct genetic diagnosis, as well as determination of the carrier status and genetic risk in family members.

Small mutations in Duchenne/Becker muscular dystrophy in 164 unrelated Polish patients.

In the 164 patients with Duchenne/Becker muscular dystrophy, we found 142 different small mutations including 51 novel mutations not listed in the LOVD, the UMD-DMD, the ClinVar, and the HGMD databases. Among all mutations, nonsense mutations occurred in 45.7%, frameshift mutations in 32.9%, and splicing mutations in 19.5%. Small mutations were distributed throughout the whole dystrophin gene. Splicing mutations were twice more common in BMD patients than in DMD patients. Eighty-two percent of mothers of the males affected with DMD/BMD were found to be carriers of small mutations.

Twin pregnancies discordant for digynic triploidy - A case series.

OBJECTIVE: To analyse natural course and perinatal management in twin pregnancies discordant for digynic triploidy. CASE REPORT: We present five cases of twins discordant for digynic triploidy. Pregnancy outcome was known for three of them. In one case, premature rupture of membranes occurred at 20 gestational weeks and both fetuses were miscarried. In two other pregnancies healthy co-twins were born at term after the triploid fetuses demise at 28 and 37 weeks. No maternal complications were observed. CONCLUSION: Twin pregnancies discordant for triploidy poses a challenge for perinatal management. Expectant management should be considered in digynic triploid cases.

Observation of the natural course of type 3 spinal muscular atrophy: data from the polish registry of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is one of the most frequent and severe genetic diseases leading to premature death or severe motor disability. New therapies have been developed in recent years that change the natural history of the disease. The aim of this study is to describe patients included in the Polish Registry of SMA, with a focus on the course of type 3 SMA (SMA3) before the availability of disease-modifying treatments. RESULTS: 790 patients with SMA were included in the registry (173 with type 1 [SMA1], 218 with type 2 [SMA2], 393 with SMA3, and six with type 4 SMA [SMA4]), most (52%) of whom were adults. Data on SMN2 gene copy number were available for 672 (85%) patients. The mean age of onset was 5 months for SMA1, 11.5 months for SMA2, and 4.5 years for SMA3. In patients with SMA3, the first symptoms occurred earlier in those with three copies of SMN2 than in those with four copies of SMN2 (3.2 years vs. 6.7 years). The age of onset of SMA3 was younger in girls than in boys (3.1 years vs. 5.7 years), with no new cases observed in women older than 16 years. Male patients outnumbered female patients, especially among patients with SMA3b (49 female vs. 85 male patients) and among patients with SMA3 with four copies of SMN2 (30 female vs. 69 male patients). 44% of patients with SMA3 were still able to walk; in those who were not still able to walk, the mean age of immobilization was 14.0 years. Patients with SMA3a (age of onset < 3 years) and three copies of SMN2 had significantly worse prognosis for remaining ambulant than patients with SMA3b (age of onset ≥ 3 years) and four copies of SMN2. CONCLUSIONS: The Registry of SMA is an effective tool for assessing the disease course in the real world setting. SMN2 copy number is an important prognostic factor for the age of onset and ambulation in SMA3. Sex and age of disease onset also strongly affect the course of SMA. Data supplied by this study can aid treatment decisions.