[Maternal uniparental disomy 14; differential diagnosis with Prader-Willi syndrome]. / Maternale uniparentale disomie 14. In de differentiaaldiagnose bij Prader-Willi-syndroom.

BACKGROUND: Maternal uniparental disomy 14 is a rare genetic disorder in which both chromosomes 14 are maternally inherited. The disorder is characterised by neonatal hypotonia and feeding difficulties, intrauterine or later growth retardation, truncal obesity and precocious puberty. During the neonatal period its clinical phenotype shows great similarities with that of Prader-Willi syndrome. CASE DESCRIPTION: We describe two patients with dysmaturity, neonatal hypotonia and feeding difficulties who initially showed clinical signs of Prader-Willi syndrome. However, molecular testing for this disorder was normal. Some years later, additional molecular testing confirmed the diagnosis of maternal uniparental disomy 14. CONCLUSION: Maternal uniparental disomy 14 shows many phenotypic similarities with Prader-Willi syndrome. In a hypotonic neonate, molecular testing for maternal uniparental disomy 14 should therefore be considered if Prader-Willi syndrome has been excluded.

X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome.

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.

Selective karyotyping in recurrent miscarriage: are recommended guidelines adopted in daily clinical practice?

BACKGROUND: Couples with recurrent miscarriage (RM) have an increased risk of one of the partners carrying a structural chromosome abnormality. On the basis of four independent risk factors, an evidence-based model was developed, which allows limiting karyotyping to high-risk couples. The aim of this study was to assess the level of adoption of selective karyotyping, its clinical consequences and the factors at the patient and hospital level that determine adoption. METHODS: A retrospective cohort study was performed in nine Departments of Obstetrics and Gynaecology, the Netherlands, in 2006. Selective karyotyping was defined as offering karyotyping to high-risk couples and refraining from karyotyping in low-risk couples. Data were collected for risk factors as described in the model for selective karyotyping, cytogenetic results as a measure for clinical consequences, and information about determinants and costs. RESULTS: A total of 530 couples were included; 252 (48%) high-risk couples and 278 (52%) low-risk couples. Among the high-risk couples, 186 (74%) were offered karyotyping. Although not advised, karyotyping was still performed in 198 (71%) low-risk couples. Overall, selective karyotyping was offered to 50% of the couples. The main determinants for adoption of the model were maternal age, obstetric history, treatment by specialists in RM and the number of patients per centre. If selective karyotyping was adopted adequately, a potential reduction of 34% of all karyotyping tests performed is possible. CONCLUSION: Selective karyotyping is applied in only half of the couples with RM in daily practice. Implementation of selective karyotyping should be a topic of future research.

Embryonic stem cell-like cells derived from adult human testis.

BACKGROUND: Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis. METHODS: Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity. RESULTS: In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice. CONCLUSIONS: Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Phenotypic discordance upon paternal or maternal transmission of duplications of the 11p15 imprinted regions.

We report on two families in which the parental origin of duplications of the BWS imprinted regions on chromosome 11p15 influences the phenotype. In family A the transmission of a t(4; 11)(q35; p15.5) translocation results in duplication of BWSIC1 and BWSIC2. If this duplication is transmitted from the father, the extra chromosomal material has the paternal imprint. This results in overexpression of IGF2 and consequently an overgrowth phenotype. If the duplication is transmitted from the mother, the extra chromosomal material has the maternal imprint, resulting in overexpression of CDKN1C and a growth retardation phenotype. In family B an interstitial duplication of BWSIC1 results in an overgrowth phenotype when inherited from the father, similar to family A. However, no change in phenotype is observed if the duplication is transmitted through the mother suggesting that increased dosage of maternally expressed genes in the duplicated region has limited effect on the phenotype.

Inherited unbalanced structural chromosome abnormalities at prenatal chromosome analysis are rarely ascertained through recurrent miscarriage.

OBJECTIVE: To determine the mode of ascertainment of inherited unbalanced structural chromosome abnormalities detected at prenatal chromosome analysis. METHODS: From the databases of three centres for clinical genetics in the Netherlands, all cases of inherited unbalanced structural chromosome abnormalities detected at prenatal chromosome analysis in the period 1992-2000 were selected. The mode of ascertainment was identified by examining the reason for prenatal chromosome analysis and the reason for parental chromosome analysis of the first structural chromosome abnormality detected within the family. RESULTS: Totally 56 cases of inherited unbalanced structural chromosome abnormalities were detected at prenatal chromosome analysis. Only one case was ascertained through two previous miscarriages (2%). The main modes of ascertainment were a previous child with an unbalanced karyotype (48%), congenital abnormalities at ultrasound examination (20%), and advanced maternal age (9%). The remaining cases had a different mode of ascertainment. CONCLUSION: Inherited unbalanced structural chromosome abnormalities detected at prenatal chromosome analysis are rarely ascertained through two or more miscarriages.

[Risk factors for structural chromosomal abnormality in > or = 2 miscarriages, as an instrument for selective karyotyping]. / Risicofactoren voor structurele chromosoomafwijking bij > or = 2 miskramen als instrument voor selectieve karyotypering.

OBJECTIVE: To identify additional risk factors and the corresponding probability of carrying a chromosome abnormality in couples with two or more miscarriages. DESIGN: Nested case-control study. METHOD: In 6 centres for clinical genetics in the Netherlands, data were collected from couples referred for karyotyping after 2 2 miscarriages from 1992-2000. Factors influencing the probability of carrier status were examined. The corresponding probability of carrier status was calculated for the various combinations of these factors. RESULTS: In total 279 carrier couples and 428 non-carrier couples were included. 4 independent factors influencing the probability of carrier status were identified: a younger maternal age at the time of second miscarriage, a history of > or = 3 miscarriages, a history of > 2 miscarriages in a brother or sister of either partner, and a history of> 2 miscarriages in parents of either partner. The calculated probability of carrier status in couples referred for chromosome analysis after two or more miscarriages, varied between 0.5-10.2%. In 18% of couples included, the risk was found to be so low (< 2.2%), that in couples with comparable risk factors, it may not be necessary to perform karyotyping. CONCLUSION: This study demonstrated that the probability of carrier status in couples with > or = 2 miscarriages is modified by additional factors. Selective chromosome analysis would result in a more effective referral policy and therefore decrease the number of chromosome analyses and lower the costs.

Increased nuchal translucency and split-hand/foot malformation in a fetus with an interstitial deletion of chromosome 2q that removes the SHFM5 locus.

OBJECTIVES: To describe and discuss the clinical, cytogenetic and molecular findings in a fetus with the first prenatally detected interstitial deletion of chromosome 2q. CASE REPORT: A fetus with increased nuchal translucency on routine ultrasound examination at 13 weeks' gestation was found to have severe upper-limb abnormalities on follow-up ultrasound examination at 16 weeks. The pregnancy was terminated, and the autopsy revealed monodactyly of the right upper limb, oligodactyly of the left upper limb and bilateral split foot, as well as atrial and ventricular septal defects and mild facial dysmorphism. RESULTS: Cytogenetic studies and haplotype analysis of the fetus and both parents showed that the fetus carried a de novo deletion encompassing a region of about 30 Mb on the paternal chromosome 2q (karyotype 46,XX,del(2)(q24.2-q32.2)). CONCLUSION: This is the first instance of increased nuchal translucency associated with a chromosome 2q deletion. Moreover, the striking malformations affecting all four of the fetus' limbs support previous suggestions that a novel locus for split-hand/foot malformation (SHFM5) lies on chromosome 2q31.

[Invasive prenatal diagnosis in the Netherlands, 1991-2000: number of procedures, indications and abnormal results detected]. / Invasieve prenatale diagnostiek in Nederland, 1991-2000: aantallen ingrepen, indicaties, en gevonden afwijkingen.

OBJECTIVE: To provide an overview of invasive prenatal diagnosis in the Netherlands during the period 1991-2000 and to analyse potential trends. DESIGN: Retrospective. METHOD: The annual results from all 13 Dutch centres for invasive prenatal diagnosis over the period 1991-2000 were combined and described, with particular emphasis on indications, number and type of invasive procedures, and number and type of abnormal results. RESULTS: The percentage of pregnancies in which invasive prenatal diagnostics were carried out increased from 5% in 1991 to 6% in 1996 and remained at the same level until 2000. 'Maternal age' was the main reason for prenatal testing (69.2-73.3% of procedures). However, the number of pregnant women aged 36 or over increased by 69.9%. An abnormal result was found in an average of 4.7% of procedures, rising from 3.6% in 1991 to 5.4% in 2000. In 70.8% of cases with abnormal results, the pregnancy was terminated. Important trends were the relative decrease of cordocentesis (-82%) and chorionic villi biopsy (-18%) in favour of amniocentesis (+48%), and a strong decrease in the number of amniocentesis procedures on indication of increased risk of neural tube defect. CONCLUSION: The total number of invasive prenatal diagnostic procedures remained stable. However, there was an important decrease in the percentage of pregnant women aged 36 or over who underwent invasive prenatal diagnosis without previous prenatal screening.

Clinical relevance of diagnosing structural chromosome abnormalities in couples with repeated miscarriage.

BACKGROUND: The annual number of parental karyotypes in cases of repeated miscarriage is increasing gradually in The Netherlands. The efficiency of offering parental karyotyping in couples with repeated miscarriage has not been evaluated before, especially not for the group with miscarriages at advanced maternal age. METHODS: A historical cohort study and nested case-control study were conducted, including couples with at least two miscarriages. Data were retrieved from medical records and telephone interviews. The obstetric follow-up was recorded for > or =2 years after the parental chromosome analysis. Data were analysed to compare ratios of carrier/non-carrier couples in whom maternal age was > or =36 or <36 years at the second, third or fourth and more miscarriage. A projected prevalence of carrier status of a structural chromosome abnormality was calculated by extrapolating the number of included patients to the original level of the total screening population. RESULTS: Forty-one couples with carrier status of a structural chromosome abnormality and 74 couples without carrier status were included. No unbalanced offspring arose after the detection of a structural chromosome abnormality. The risk of being a carrier was not significantly lower (as might be expected) when women were > or =36 years. Ascertainment after two, three, or four and more miscarriages did not change these findings. CONCLUSIONS: Karyotyping of 1324 couples ascertained for repeated miscarriage did not yield an unbalanced fetal chromosome pattern after the ascertainment of parental carrier status. In women with advanced maternal age, the frequency of carrier status was not lower than in younger women.

Prenatal diagnosis of a karyotypically normal pregnancy in a mother with a supernumerary neocentric 13q21 -->13q22 chromosome and balancing reciprocal deletion.

An adult female patient with a history of miscarriages was found to be carrying a stable supernumerary chromosome. The patient also carried a reciprocal paracentric deletion in chromosome 13q21/22. Microdissection and reverse fluorescence in situ hybridization FISH revealed that this supernumerary chromosome was derived from region 13q21 --> 13q22. The presence of a neocentromere on this supernumerary chromosome was confirmed by the absence of detectable alpha satellite DNA using FISH and the presence of centromere proteins CENP-C and CENP-A using immunofluorescence. The absence of telomere sequences suggests that the marker is a ring chromosome (r(13)). FISH using ordered BACs from the chromosome region 13q21 --> 13q31 permitted the precise positioning of the r(13) chromosome and the corresponding deletion to chromosome bands 13q21.32 --> 13q22.2. BAC 280J7 from within the r(13) was used as a FISH probe for the prenatal analysis of amniocytes at 16 weeks of gestation, which revealed a normal karyotype for the fetus. This r(13) chromosome represents the first description of chromosome 13 of the rarer class of neocentric chromosomes that are derived from interstitial deletions. It represents the first example of prenatal diagnosis in a phenotypically normal female that was ascertained to carry a neocentric marker. The presence of such a neocentric marker/deletion karyotype in a parent presents unique possible karyotypic outcomes for conceptions and unusual challenges for genetic counseling.

FISH analysis of fetal nucleated red blood cells from CVS washings in cases of aneuploidy.

OBJECTIVE: In chorionic villus sampling (CVS) the chromosome analysis is inconclusive in 1-2% of the samples. In many cases follow-up amniocentesis is performed. Fetal nucleated red blood cells (FNRBCs) are present in washings of chorionic villus samples. We wanted to establish whether analysis of these true fetal cells, using fluorescence in situ hybridization (FISH), could support the CVS karyotype. METHODS: We analysed washings of first trimester chorionic villi from non-mosaic 45,X (n=6) and full trisomy 18 cases (n=7). FNRBCs were identified by immunostaining and FISH was performed with chromosome-specific probes for X, Y and 18. RESULTS: In all 13 samples FNRBCs were present (between 4 and 30 cells per sample). Five cases of monosomy X showed one X signal in 89-100% of the nuclei; in the other case 50% of the nuclei displayed one signal. In the trisomy 18 cases three spots were seen in 60-100% of the cells. CONCLUSION: The CVS aneuploidy was confirmed in FNRBCs in all samples, so FISH on FNRBCs can be used in cases of non-mosaic numerical chromosomal abnormalities. This test can confirm a CVS diagnosis of monosomy X or trisomy 18 and thus minimize the risk for false-positive diagnoses. An additional invasive test may be prevented.

Genetic analysis of fetal nucleated red blood cells from CVS washings.

Chorionic villus sampling (CVS) is an established invasive prenatal diagnostic method for the detection of fetal chromosome aberrations. In 1-2% the karyotype result of CVS is inconclusive and follow-up confirmation will be required. To avoid another invasive procedure we examined fetal nucleated red blood cells (NRBCs) from CVS washings for genetic analysis. We analysed the washings of 20 chorionic villi samples of male fetuses. Fetal NRBCs were immunostained by an antibody against embryonic haemoglobin (HbE). FISH was performed with probes specific for the X and Y chromosome and the nucleus was counterstained with DAPI. Cells positive for the antibody, as well as for DAPI, were collected and stored by a semi-automated microscope. An operator reviewed those cells for their FISH signals. In 19 out of 20 CVS washings we found nucleated cells positive for HbE together with XY FISH signals. In none of the washings HbE positive cells with two X signals were found. Our results indicate that anti-HbE is a very specific antibody for identifying fetal NRBCs. NRBCs from CVS washings can be used as an additional fetal tissue for first trimester prenatal diagnosis.

The outcome of pregnancies with confined placental chromosome mosaicism in cytotrophoblast cells.

Cytogenetic findings and outcome of pregnancy are reported in 108 cases in which confined placental mosaicism (CPM, n = 101) or generalized mosaicism (n = 7) was found at or after first-trimester chorionic villus sampling. In all samples, a (semi)direct cytogenetic analysis of cytotrophoblast cells was performed. Two pregnancies with CPM ended in a spontaneous abortion before 28 weeks (1.9 per cent). In 15 cases the pregnancy was terminated: eight cases were shown to be examples of CPM; seven cases can be considered as examples of generalized mosaicism. A normal cytogenetic result was obtained after follow-up amniocentesis in 88 of the remaining 91 cases. In three cases, no amniocentesis was performed but confirmation of a normal karyotype was obtained in other cells. One of the 91 pregnancies was nevertheless terminated for psychosocial reasons. One child died perinatally and another on the seventh day after birth. The birth weight is known for 89 children; the curve shows a normal distribution. In 11 of these children (12.3 per cent), the birth weight was found to be below the tenth centile. The outcome in a subgroup of eight pregnancies with CPM and involvement of chromosome 13, 16, or 22, however, revealed two fetal losses and four children with a birth weight below the tenth centile (75 per cent).

An evaluation of 75 terminations of pregnancy based on abnormal laboratory findings at first trimester CVS.

Seventy-five selective terminations, based on abnormal laboratory findings at first-trimester CVS, were performed in 1581 consecutive pregnancies. In all cases a (semi-) direct method of cytogenetic analysis was used. The 75 abortions were analysed in number of ways. Confirmatory studies showed that three cases had to be considered as false-positive findings, and in one other case the results were inconclusive. Based on literature data, it was estimated that 41 of the 75 pregnancies would have resulted in seriously handicapped children, surviving beyond the age of 1 year, if no termination of pregnancy had taken place. Negative side-effects of the procedure include: spontaneous abortion of chromosomally normal fetuses due to the CVS procedure itself and the need for a number of secondary amniocenteses (5.1%). The advantage of DNA diagnosis in X-linked diseases is illustrated by comparing the CVS results with a previously published amniocentesis study.

PIP: 75 selective terminations, based on abnormal laboratory findings at 1st trimester CVS, were performed in 1581 consecutive pregnancies. In all cases, a semidirect method of cytogenetic analysis was used. The 75 abortions were analyzed in many ways. Confirmatory studies showed that 3 cases were considered false-positive findings, and in 1 other case, the results were inconclusive. Based on literature data, it was estimated that 41.75 pregnancies would have resulted in seriously handicapped children, surviving beyond 1 year of age, if no pregnancy termination had taken place. Negative side effects of the procedure include: spontaneous abortion of chromosomally normal fetuses due to the CVS procedure itself and the need for a number of secondary amniocenteses (5.1%). The advantage of DNA diagnosis in x-linked diseases is illustrated by comparing the CVS results with a previously published amniocentesis study.