Peter Miny

Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood

OBJECTIVE We wanted to test whether the recently described method of using the transferrin receptor system for fluorescence-activated cell-sorter enrichment of nucleated red blood cells can be used for prenatal diagnosis from maternal blood. STUDY DESIGN Instead of the laborious, expensive fluorescence-activated cell-sorter system, we used the newly described magnetic-activated cell sorter. RESULTS An effective enrichment could be achieved with separation of lymphocyte subsets. With the transferrin receptor, however, the enrichment was very inefficient because of the poor specificity of the antibody itself. Even in umbilical cord blood only 25% of nucleated red blood cells were labeled as demonstrated by immunogold silver enhancement of transferrin receptor-labeled cells. CONCLUSION In spite of the availability of a fast and effective separation method (magnetic-activated cell sorter) the use of the transferrin receptor antigen alone is not likely to enable a reliable identification of fetal cells in maternal circulation.

The mutation spectrum in RECQL4 diseases.

Mutations in the RECQL4 gene can lead to three clinical phenotypes with overlapping features. All these syndromes, Rothmund–Thomson (RTS), RAPADILINO and Baller–Gerold (BGS), are characterized by growth retardation and radial defects, but RAPADILINO syndrome lacks the main dermal manifestation, poikiloderma that is a hallmark feature in both RTS and BGS. It has been previously shown that RTS patients with RECQL4 mutations are at increased risk of osteosarcoma, but the precise incidence of cancer in RAPADILINO and BGS has not been determined. Here, we report that RAPADILINO patients identified as carriers of the c.1390+2delT mutation (p.Ala420_Ala463del) are at increased risk to develop lymphoma or osteosarcoma (6 out of 15 patients). We also summarize all the published RECQL4 mutations and their associated cancer cases and provide an update of 14 novel RECQL4 mutations with accompanying clinical data.

Parental origin and mechanisms of formation of cytogenetically recognisable de novo direct and inverted duplications

Cytogenetic, FISH, and molecular results of 20 cases with de novo tandem duplications of 18 different autosomal chromosome segments are reported. There were 12 cases with direct duplications, three cases with inverted duplications, and five in whom determination of direction was not possible. In seven cases a rearrangement between non-sister chromatids (N-SCR) was found, whereas in the remaining 13 cases sister chromatids (SCR) were involved. Paternal and maternal origin (7:7) was found almost equally in cases with SCR (3:4) and N-SCR (4:3). In the cases with proven inversion, there was maternal and paternal origin in one case each. Twenty three out of 43 cytogenetically determined breakpoints correlated with common or rare fragile sites. In five cases, including all those with proven inverse orientation, all breakpoints corresponded to common or rare fragile sites. In at least two cases, one with an interstitial duplication (dup(19)(q11q13)) and one with a terminal duplication (dup(8) (p10p23)), concomitant deletions (del(8) (p23p23.3) and del(19)(q13q13)) were found.

Low rates of pregnancy termination for prenatally diagnosed Klinefelter syndrome and other sex chromosome polysomies

Over the past 9 years we counseled 55 couples whose unborn child was found to carry a sex chromosome polysomy. We performed a survey of postcounseling parental decisions about continuation or termination of these pregnancies. Of the 55 embryos or fetuses, 23 had the karyotype 47,XXY, 10 had 47,XYY, and 12 had 47,XXX. In addition, there were 10 instances of true mosaicism, i.e. 47,XXY/46,XY (n = 5), 47,XYY/46,XY (n = 2), or 47,XXX/46,XX (n = 3). Mean gestational age (+/-standard deviation) at diagnosis was 18.3+/-3.0 weeks. After comprehensive genetic counseling 48 (87.3%) of these pregnancies were carried to term. In seven cases (12.7%) the parents elected a pregnancy termination. Two of 31 pregnancies (6.5%) primarily ascertained at our center were aborted, whereas amongst the 24 referred cases, 5 couples (20.8%) opted for a termination. The mean gestational age of the terminated pregnancies was 19.7 weeks. The overall termination rate of 12.7% appears low in comparison with literature data. Most reports from other institutions present termination rates between 32 and 66%. The reason for the low rate of induced abortions in our study cohort is not clear. Cultural differences in parental perception of sex chromosomal polysomies may be of importance, and peculiarities of genetic counseling at our institution could also play a role. Although counseling was nondirective, we did put emphasis on providing prospective parents with information from unbiased follow-up studies of children with Klinefelter syndrome and other sex chromosome polysomies.

Deletion in Xp22.11: PTCHD1 is a candidate gene for X-linked intellectual disability with or without autism

Filges I, Röthlisberger B, Blattner A, Boesch N, Demougin P, Wenzel F, Huber AR, Heinimann K, Weber P, Miny P. Deletion in Xp22.11: PTCHD1 is a candidate gene for X‐linked intellectual disability with or without autism.

Reduced expression by SETBP1 haploinsufficiency causes developmental and expressive language delay indicating a phenotype distinct from Schinzel–Giedion syndrome

Background Mutations of the SET binding protein 1 gene (SETBP1) on 18q12.3 have recently been reported to cause Schinzel–Giedion syndrome (SGS). As rare 18q interstitial deletions affecting multiple genes including SETBP1 correlate with a milder phenotype, including minor physical anomalies and developmental and expressive speech delay, mutations in SETBP1 are thought to result in a gain-of-function or a dominant-negative effect. However, the consequence of the SETBP1 loss-of-function has not yet been well described. Methods Microarray-based comparative genomic hybridisation (aCGH) analyses were performed to identify genetic causes for developmental and expressive speech delay in two patients. SETBP1 expression in fibroblasts obtained from one of the patients was analysed by real-time RT-PCR and western blotting. A cohort study to identify nucleotide changes in SETBP1 was performed in 142 Japanese patients with developmental delay. Results aCGH analyses identified submicroscopic deletions of less than 1 Mb exclusively containing SETBP1. Both patients show global developmental, expressive language delay and minor facial anomalies. Decreased expression of SETBP1 was identified in the patients skin fibroblasts. No pathogenic mutation of SETBP1 was identified in the cohort study. Conclusion SETBP1 expression was reduced in a patient with SETBP1 haploinsufficiency, indicating that the SETBP1 deletion phenotype is allele dose sensitive. In correlation with the exclusive deletion of SETBP1, this study delimits a milder phenotype distinct from SGS overlapping with the previously described phenotype of del(18)(q12.2q21.1) syndrome including global developmental, expressive language delay and distinctive facial features. These findings support the hypothesis that mutations in SETBP1 causing SGS may have a gain-of-function or a dominant-negative effect, whereas haploinsufficiency or loss-of-function mutations in SETBP1 cause a milder phenotype.

Ratio of fetal to maternal DNA is less than 1 in 5000 at different gestational ages in maternal blood.

Using Southern hybridization with the DNA probe pY3.4, we were not able to detect fetal DNA in blood of 36 pregnant women carrying male fetuses. Gestational ages ranged from 8–40 weeks of pregnancy. Using the same DNA probe, we were able to detect the male‐specific signal in experimental dilution series down to 1/5000 on autoradiograms. We conclude that the ratio of fetal DNA in maternal circulation, in contrast to previous estimations, must be lower than 1/5000.

Prenatal diagnosis and management of fetal hydrocephaly and lissencephaly

Two cases of prenatal diagnosis of lissencephaly are presented in the context of a series of 118 cases of prenatally diagnosed hydrocephalus. Within this series there was one case of Walker-Warburg syndrome and another of Miller-Dieker syndrome. It is stressed that the cases reported here of ventriculomegaly diagnosed in utero show a very different outcome from those in published studies of fetal hydrocephalus which only deal with patients in whom the diagnosis was determined after birth. In those postnatal series there is a considerable selection bias, and the fate of the fetuses reported here was much worse than in postnatal series. Of the 118 fetuses 6 had fetal infections, 6 had chromosomal abnormalities, 26 had associated spina bifida, 64 fetuses had associated other anomalies, and only 28 had isolated hydrocephalus. Although it is difficult to determine the prognosis individually after prenatal diagnosis of ventriculomegaly, the data presented here may be helpful in counseling parents prenatally. The counseling should be performed with the collaboration of obstetricians, pediatricians, surgeons, and geneticists.

Benefits of placental biopsies for rapid karyotyping in thesecond and third trimesters (late chorionic villus sampling) in high-risk pregnancies

In a total of 301 placental biopsies in the second and third trimesters, 225 were performed because of suspicious ultrasonographic findings. In this group there was a high rate of aneuploidies (20%). As opposed to the alternative methods for rapid karyotyping, placental biopsies can easily be performed even in pregnancies with abnormal amounts of amniotic fluid. Oligohydramnios and polyhydramnios were key ultrasonographic findings in 38% of cases and were found to be associated with 22% of abnormal chromosomal findings. Suggestive ultrasonographic findings seem to justify the exclusive use of direct preparation.

Molecular basis of β-thalassemia in Turkey: detection of rare mutations by direct sequencing

SummaryUsing restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for β-thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.