Preoperative joint line convergence angle correction is a key factor in optimising accuracy in varus knee correction osteotomy.

PURPOSE: This study aimed to identify and prevent preoperative factors that can be influenced in preoperative planning to reduce postoperative malcorrections. METHODS: The method used in this study was a retrospective two-centre analysis of 78 pre and postoperative fully weight-bearing radiographs of patients who underwent valgus osteotomy correction due to symptomatic medial compartment osteoarthritis. A computer software (TraumaCad®) was used to aim for an intersection point of the mechanical tibiofemoral axis (mTFA) with the tibia plateau at 55-60% (medial = 0%, lateral = 100%). Postoperative divergence ± 5% of this point was defined as over- and undercorrection. Preoperative joint geometry factors were correlated with postoperative malcorrection. Planning was conducted using the established method described by Miniaci (Group A) and with additional correction of the joint line convergence angle (JLCA) using the formula JLCA-2/2 (Group B). Additionally, in a small clinical case series, planning was conducted with JLCA correction. Statistical analysis was performed using (multiple) linear regression analysis and analysis of variance (ANOVA) with p < 0.05 considered significant. RESULTS: In 78 analysed cases, postoperative malcorrection was detected in 37.2% (5.1% undercorrection, 32.1% overcorrection). Linear regression analysis revealed preoperative body mass index (BMI, p = 0.04), JLCA (p = 0.0001), and osteotomy level divergence (p = 0.0005) as factors correlated with overcorrection. In a multiple regression analysis, JLCA and osteotomy level divergence remained significant factors. Preoperative JLCA correction reduced the planned osteotomy gap (A 9.7 ± 2.8 mm vs B 8.3 ± 2.4 mm; p > 0.05) and postoperative medial proximal tibial angle (MPTA: A 94.3 ± 2.1° vs B 92.3 ± 1.5°; p < .05) in patients with preoperative JLCA ≥ 4°. The results were validated using a virtual postoperative correction of cases with overcorrection. A case series (n = 8) with a preoperative JLCA > 4 revealed a postoperative accuracy using the JLCA correction of 3.4 ± 1.9%. CONCLUSION: Preoperative JLCA ≥ 4° and tibial osteotomy level divergence were identified as risk factors for postoperative overcorrection. Preoperative JLCA correction using the formula JLCA-2/2 is proposed to better control ideal postoperative correction and reduce MPTA. The intraoperatively realised osteotomy level should be precisely in accordance with preoperative planning. LEVEL OF EVIDENCE: III, cross-sectional study.

Branchio-otic syndrome caused by a genomic rearrangement: clinical findings and molecular cytogenetic studies in a patient with a pericentric inversion of chromosome 8.

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype.

A family with an inverted tandem duplication 5q22.1q23.2.

Here, we report a 3-year-old boy with short stature, developmental delay and mild facial dysmorphic signs. Karyotype analysis and array-CGH revealed a pure duplication 5q22.1q23.2 with a length of 14.25 Mb. As demonstrated by multicolor-fluorescence in situ hybridization, the duplicated segment was orientated in an inverted tandem manner. One of the 2 older half-brothers of the index patient was intellectually disabled and showed short stature as well. The mother of the siblings was only 149 cm in height. The affected half-brother as well as the mother of the siblings were tested positive for the same duplication. Duplications of the long arm of chromosome 5 are rare. There are 16 reported cases of different 5q segments with a pure duplication and no additional chromosomal imbalance. In order to refine the 5q-duplication phenotype, reported cases were recently classified in 3 groups on the basis of clinical findings and the involved chromosome segments. However, our case does not fit in any of these groups but is placed in the interjacent chromosomal area between 2 of these groups. Overall, this is the second reported family with a duplication of 5q22.1q23.2 and both families share phenotypic features like short stature, facial dysmorphic signs and speech delay. The reported family provides further information for delineating phenotype-genotype correlations of pure duplications of the 5q region.

Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants?

Array comparative genomic hybridization (array CGH) is now widely adopted as a first-tier clinical diagnostic test in individuals with unexplained developmental delay/intellectual disability (DD/ID) and congenital anomalies. Our study aimed at enlarging the phenotypic spectrum associated with clinically relevant copy number variants (CNVs) as well as delineating clinical criteria, which may help separating patients with pathogenic CNVs from those without pathogenic CNVs. We performed a retrospective review of clinical and array CGH data of 342 children with unexplained DD/ID. The phenotypic features of patients with clinically significant CNV were compared with those without pathogenic CNVs. Array CGH detected pathogenic CNVs in 13.2% of the patients. Congenital anomalies, especially heart defects, as well as primary microcephaly, short stature and failure to thrive were clearly more frequent in children with pathogenic CNVs compared with children with normal array CGH results. Thus, we assume that in patients with unexplained DD/ID, array CGH will more probably detect a significant CNV if any of these features is part of the patient's phenotype.

Ring chromosome 22 and neurofibromatosis type II: proof of two-hit model for the loss of the NF2 gene in the development of meningioma.

Carriers of a ring chromosome 22 are mentally retarded and show variable facial dysmorphism. They may also present with features of neurofibromatosis type II (NF2) such as vestibular schwannomas and multiple meningiomas. In these cases, tumourigenesis has been suspected to be caused by the loss of both alleles of the NF2 gene, a tumour suppressor localized in 22q12.2. Here, we describe an 18-year-old patient with constitutional ring chromosome 22 and mental retardation who developed rapid-onset spastic paraparesis at the age of 15 years. The causative spinal meningioma at the level of T3, which compressed the spinal cord, was surgically removed, and the patient regained ambulation. Array comparative genomic hybridization (array CGH) and multiplex ligation-dependent probe amplification (MLPA) analyses in blood revealed a terminal deletion in 22q13.32, not comprising the NF2 gene. In tumour tissue, loss of the whole ring chromosome 22 including one NF2 gene due to mitotic instability constituted the likely first hit, while a point mutation in the other allele of the NF2 gene (c.784C>T, p.R262X) was shown as second hit. We review all cases from the literature and suggest clinical guidelines for surveillance of patients with ring chromosome 22.

Is there a yet unreported unbalanced chromosomal abnormality without phenotypic consequences in proximal 4p?

Unbalanced chromosomal abnormalities (UBCA) are reported for >50 euchromatic regions of almost all human autosomes. UBCA are comprised of a few megabases of DNA, and carriers are in many cases clinically healthy. Here we report on a partial trisomy of chromosome 4 of the centromere-near region of the short arm of chromosome 4 present as a small supernumerary marker chromosome (sSMC). The sSMC was present in >70% of amnion cells and in 60% of placenta. Further delineation of the size of the duplicated region was done by molecular cytogenetics and array comparative genomic hybridization. Even though the sSMC lead to a partial trisomy of ~9 megabase pairs, a healthy child was born, developing normally at 1 year of age. No comparable cases are available in the literature. Thus, we discuss here the possibility of having found a yet unrecognized chromosomal region subject to UBCA.

Forty-eight new cases with infertility due to balanced chromosomal rearrangements: detailed molecular cytogenetic analysis of the 90 involved breakpoints.

A molecular cytogenetic study was performed on 48 infertile patients who were identified as carriers of balanced translocations (40 cases), inversions (6 cases) or insertions (2 cases) by means of banding cytogenetics. Cases with a Robertsonian translocation or pericentric inversion 2 or 9 were not included. In summary, 100 break-events occurred in these patients, and 90 different chromosomal regions were involved. Thus, this study confirmed the presence of abnormal karyotypes in a subgroup of patients seeking infertility treatment. Breaks were demonstrated to appear preferentially in GTG-light bands in these patients. Furthermore, the observed breakpoints were associated with genomic regions prone to instability due to the presence of segmental duplications. Nonetheless, further detailed molecular analysis will be necessary in the future to characterize the mechanisms and genetic basis for this phenomenon.

A simple and inexpensive high density dialysis tubing cell culture system for the in vitro production of monoclonal antibodies in high concentration.

This paper describes the construction and application of a low-cost roller bottle-like culture appliance in which hybridoma cells can be cultivated in high density in dialysis tubing. The appliance facilitates the simultaneous culture of up to four cell lines yielding 50 ml culture volume of each. Samples for follow-up analysis of the cultures can easily be taken when needed through sample ports. In order to obtain high cell densities (at least 10(7) cells/ml), high cell viability (at least 50%) and high antibody yield (at least 1.0 mg/ml) the bottle is rolled at a speed of 4-6 rpm and is gassed continuously by a micropump driven by rechargeable NiCd batteries fixed to the culture flask. Depending on the individual properties of the hybridoma lines tested, the cells may be cultured for 1-2 weeks, and cell densities of up to 30 x 10(6) cells/ml with viabilities of approximately 50% and monoclonal antibodies in concentrations of up to 2.8 mg/ml may be obtained. In their properties the monoclonal antibodies produced by this in vitro procedure are indistinguishable from those prepared in the form of conventional stationary culture supernatant or of ascitic fluid. Specific antibody content is within the same range as in ascitic fluid. Consequently, the monoclonal antibodies can be purified in one step, e.g., by ion exchange chromatography from the culture supernatant. Therefore, the newly developed culture device and the culture method described is a useful alternative to ascites production in live mice.

In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter.

This paper describes a new and easy to handle reusable minifermenter for high-density culture of hybridoma and other cells. The culture apparatus is composed of two modules: a 40 ml disposable cell culture and antibody production chamber (the 'production module') and a 550 ml medium reservoir (the 'supply module'). The two modules are separated from each other by a dialysis membrane allowing passage of low molecular mass nutrients and metabolites. The monoclonal antibodies are produced and enriched in the production module. The outer part of this module is made from a thin gas-permeable silicone rubber membrane allowing exchange of gases (oxygen and carbon dioxide). To start the culture, the cells are injected into the production module through ports in the silicone rubber which are equipped with Luer Lock connectors. Samples can be removed in the same way. For culturing, the minifermenter is rolled on a roller apparatus in a carbon dioxide-supplied incubator. Depending on the individual properties of the hybridoma cells cultured, cell densities of more than 10 x 10(6) (in some cases up to 35 x 10(6)) cells per ml and monoclonal antibody concentrations of several mg per ml can be obtained in the new minifermenter. On average, 61 mg (range: 9-159 mg) could be produced within 1-4 weeks. In terms of their properties the monoclonal antibodies produced in the new modular minifermenter were indistinguishable from antibodies prepared from ascitic fluid or from the supernatant of conventional stationary culture. The culture method is a useful alternative to the in vivo production method in mice. In addition, it represents a completely new, inexpensive and easy to handle general solution to the problem of culturing cells in high density and obtaining cellular products in high concentrations.

Supernumerary small marker chromosome (SMC) and uniparental disomy 22 in a child with confined placental mosaicism of trisomy 22: trisomy rescue due to marker chromosome formation.

Trisomy rescue is one of various proposed mechanisms in formation of supernumerary small marker chromosomes (SMC) and uniparental disomy (UPD). In the present report a small de novo marker chromosome derived from chromosome 14 or 22 was diagnosed at prenatal diagnosis due to maternal age. Follow up investigations at birth revealed mosaicism 47,XX,+mar/46,XX. Using FISH, the marker was positive for the probe D14/22Z1, but negative for the probes midi 54 and D22Z4. Using three informative markers both chromosomes 22 were shown to be inherited from the mother (UPDmat). The results are consistent with nondisjunction at maternal meiosis I. The girl is 18 months old now and phenotypically normal. Cardiac and abdominal malformations were excluded by sonographic examinations. Motor and mental development is according to or ahead of developmental milestones (free walking with 10 months, first words at 12 months). The case confirms that maternal UPD 22 most likely is not associated with clinical abnormalities. According to FISH results, UPD 22, and 47,XX,+22 in the placenta, we conclude that the SMC was derived from alpha satellite sequences of chromosome 22. This case for the first time gives evidence that early postzygotic reduction of a chromosome to a small marker chromosome is a real existing mechanism to rescue a conceptus with trisomy.

Chromosomal mosaicism of trisomy 7 restricted to chorionic villi.

Chromosomal mosaicism confined to the placenta is a serious problem in first-trimester fetal diagnosis. We report a case of mosaicism of trisomy 7. The aneuploid cell line could not be confirmed in fetal tissue.

Maternal serum hCG and SP1 in pregnancies with fetal aneuploidy.

In a retrospective study, maternal serum levels of chorionic gonadotropin (hCG) and pregnancy specific beta 1-glycoprotein (SP1) from 63 pregnancies with aneuploid fetuses were compared to the levels observed in pregnancies with a chromosomally normal fetus. Thirty-eight percent of the abnormal pregnancies had elevated levels (greater than 2.0 multiples of the normal median [MoM]) of hCG and 14% had depressed levels (less than 0.25 MoM). With a false-positive rate of 5%, 44% of the 42 fetuses with trisomy 21 would have been detected by elevated hCG levels. With the same false-positive rate, only 21% had elevated SP1 levels. hCG was significantly depressed in 12 pregnancies affected by fetal trisomy 18.

Down syndrome at birth not detected by first-trimester chorionic villus sampling.

A case of a false-negative first-trimester diagnosis following chorionic villus sampling is reported that ended with the birth of a child with Down syndrome. Chromosome analysis of 30 metaphases from 24 h-cultured chorionic villi obtained in week 12 of gestation showed a normal chromosome constitution. However, the newborn showed manifestations of Down syndrome, and 99 of 100 metaphases analysed from cultured lymphocytes showed 47, XY, + 21. The remaining metaphase was normal (46, XY).

First confirmed case with paternal uniparental disomy of chromosome 16.

The existence of paternal uniparental disomy of chromosome 16 [upd(16)pat] has previously been suspected but has not been proven. We report prenatal detection and follow-up of isodisomic upd(16)pat in a child with minimal defects but otherwise normal development. Our results indicate that isodisomic upd(16)pat is associated with a normal outcome if no recessive mutation is reduced to homozygosity.

Additional dark G-band in the p-arm of chromosome 19 due to a paracentric inversion with a breakpoint in the pericentromeric heterochromatin.

Paracentric inversions in chromosome 19 have rarely been described. Here we present an inv(19)(p11p13.1) with a breakpoint in the pericentromeric heterochromatin which leads to an additional dark G-band in the p-arm of chromosome 19. The rearranged chromosome segregated in two generations of a family without any phenotypic effects. A detailed characterization of the inv(19) by molecular cytogenetic techniques is presented.

Short stature in a mother and daughter caused by familial der(X)t(X;X)(p22.1-3;q26).

Deletions of the terminal Xp regions, including the short-stature homeobox (SHOX) gene, were described in families with hereditary Turner syndrome and Léri-Weill syndrome. We report on a 10-2/12-year-old girl and her 37-year-old mother with short stature and no other phenotypic symptoms. In the daugther, additional chromosome material was detected in the pseudoautosomal region of one X chromosome (46,X,add(Xp.22.3)) by chromosome banding analysis. The elongation of the X chromosome consisted of Giemsa dark and bright bands with a length one-fifth of the size of Xp. The karyotype of the mother demonstrated chromosome mosaicism with three cell lines (46,X,add(X)(p22.3) [89]; 45,X [8]; and 47,X,add(X)(p22.3), add(X)(p22.3) [2]). In both daughter and mother, fluorescence in situ hybridization (FISH), together with data from G banding, identified the breakpoints in Xp22.1-3 and Xq26, resulting in a partial trisomy of the terminal region of Xq (Xq26-qter) and a monosomy of the pseudoautosomal region (Xp22.3) with the SHOX gene and the proximal region Xp22.1-3, including the steroidsulfatase gene (STS) and the Kallmann syndrome region. The derivative X chromosome was defined as ish.der(X)t(X;X)(p22.1-3;q26)(yWXD2540-, F20cos-, STS-, 60C10-, 959D10-, 2771+, cos9++). In daughter and mother, the monosomy of region Xp22.1-3 is compatible with fertility and does not cause any other somatic stigmata of the Turner syndrome or Léri-Weill syndrome, except for short stature due to monosomy of the SHOX gene.

Prenatal diagnosis of the Pallister-Killian mosaic aneuploidy syndrome by CVS.

Prenatal cytogenetic analysis at 11 weeks of gestation revealed an abnormal karyotype 47,XX,+mar in all metaphases obtained from a chorionic villi sample after 24 h culture. Karyotyping of amniotic fluid cells in the second trimester showed mosaicism 47,XX,+i(12p)/46,XX with 10% aneuploid cells. The pregnancy was terminated at 20 weeks of gestation on the patient's request. The aborted fetus showed typical manifestations of the Pallister-Killian mosaic aneuploidy syndrome. The identity of the supernumerary isochromosome 12p was proven by LDH isozyme electrophoresis using cultured fibroblasts and by nonradioactive in situ hybridization using a biotinylated set of chromosome 12-specific DNA probes.

Felbamate and meprobamate: a comparison of their anticonvulsant properties.

The anticonvulsant effect of felbamate and meprobamate were compared in a series of models for seizure activity and regarding their neurotoxic action. In the MES test, felbamate was active below neurotoxic doses. Meprobamate had an ED50 in the range of neurotoxic doses. The s.c. PTZ test was influenced by meprobamate in a fairly low dosage (ED50 66 mg/kg), but for felbamate no clearly dose-related effect could be shown up to 150 mg/kg. Reflex epilepsy in gerbils was stronger suppressed by meprobamate (ED50 34 mg/kg) than by felbamate (ED50 63 mg/kg). In amygdala kindled rats, meprobamate proved to be the most active compound, both regarding treatment of fully kindled rats, development of kindling and independent discharges from a mirror focus (secondary epileptogenesis), which were fully suppressed by oral treatment with 80 mg/kg for 30 days. Both drugs were weakly effective in a model for absence epilepsy in rats. The unexpectedly high activity of meprobamate justifies a second look at the anticonvulsant properties of the drug, especially since it was extensively used as an anxiolytic drug in the past with few obvious serious side effects.

A phenotypically normal liveborn male after prenatal diagnosis of trisomy 20 mosaicism.

We report on a case of prenatally diagnosed true trisomy 20 mosaicism in amniocytes. Cytogenetic analysis was performed postnatally on lymphocytes and extra-embryonic tissues. For analysing uroepithelial cells we established a new cell nuclei preparation protocol for FISH (Fluorescence In Situ Hybridization). Trisomy 20 cells could not be confirmed after birth. The origin or trisomy 20 cells in amniotic fluid remains unclear. The phenotypically normal male baby is developing normal.