vhnf1 integrates global RA patterning and local FGF signals to direct posterior hindbrain development in zebrafish.

The vertebrate hindbrain is transiently divided along the anterior-posterior axis into seven morphologically and molecularly distinct segments, or rhombomeres, that correspond to Hox expression domains. The establishment of a proper 'hox code' is required for the development of unique rhombomere identities, including specification of neuronal fates. valentino (val), the zebrafish ortholog of mafB/Kreisler (Kr), encodes a bZip transcription factor that is required cell autonomously for the development of rhombomere (r) 5 and r6 and for activation of Hox group 3 gene expression. Recent work has demonstrated that the expression of val itself depends on three factors: retinoic acid (RA) signals from the paraxial mesoderm; fibroblast growth factor (Fgf) signals from r4; and variant hepatocyte nuclear factor 1 (vhnf1, also known as tcf2), a homeodomain transcription factor expressed posterior to the r4-5 boundary. We have investigated the interactions between these inputs onto val expression in the developing zebrafish hindbrain. We show that RA induces val expression via activation of vhnf1 expression in the hindbrain. Fgf signals from r4, acting through the MapK pathway, then cooperate with Vhnf1 to activate val expression and subsequent r5 and r6 development. Additionally, vhnf1 and val function as part of a multistep process required for the repression of r4 identity in the posterior hindbrain. vhnf1 acts largely independently of val to repress the r4 'hox code' posterior to the r4-5 boundary and therefore to block acquisition of r4-specific neuronal fates in the posterior hindbrain. However, vhnf1 is not able to repress all aspects of r4 identity equivalently. val is required downstream of vhnf1 to repress r4-like cell-surface properties, as determined by an 'Eph-ephrin code', by repressing ephrin-B2a expression in r5 and r6. The different requirements for vhnf1 and val to repress hoxb1a and ephrin-B2a, respectively, demonstrate that not all aspects of an individual rhombomere's identity are regulated coordinately.

Maternal uniparental disomy 16 and genetic counseling: new case and survey of published cases.

Uniparental disomy (UPD) is the occurrence of both homologous chromosomes from one parent. Maternal UPD(16) is the most often reported UPD other than UPD(15); almost all cases are associated with confined placental mosaicism (CPM). Most of maternal UPD(16) cases are characterised by intrauterine growth retardation (IUGR) and different congenital malformations. Maternal UPD(16) has therefore been suspected to have clinical effects: however, the lack of uniqueness and specificity of the birth defects observed suggests that the phenotype may be related in parts to placental insufficiency. We report on a new case of maternal UPD(16) associated with low level trisomy 16 mosaicism in placenta and fetus. IUGR was noticed at 19 gestational weeks and the fetus died intrauterine. Apart from different craniofacial dysmorphisms she showed anal atresia. While IUGR is probably associated with trisomy 16 mosaicism, anal atresia is more characteristic for maternal UPD( 16). Considering the features in our patient as well as those in maternal UPD (16) cases from the literature, indications for UPD (16) testing can be defined: They include trisomy 16 mosaicism, IUGR and congenital anomalies (anal atresia, congenital heart defects). However, there is an overlap of clinical signs in mosaic trisomy 16 cases mosaic for maternal UPD(16) as opposed to those mosaic for biparental disomy 16. The management of trisomy 16 pregnancies should not differ from those in which maternal UPD(16) is confirmed. Therefore, a prenatal testing for UPD(16) is not useful, but it should be offered postnatally. The molecular genetic proof of maternal UPD(16) excludes an increased recurrence risk for the family for further pregnancies.

[Intersexuality in dogs: causes and genetics]. / Intersexualität beim Hund: Ursachen und Genetik.

Failures in the establishment of chromosomal, gonadal and phenotypic sex can cause intersexuality in dogs. Thus, diagnosis of chimaerism, mosaicism, sex reversal syndrome, and male or female pseudohermaphroditism in intersex individuals has to be based on the inspection of the chromosomes, gonads and the phenotypic appearance of the reproductive organs. In a study over two years, seven dogs of different breeds suspected to be intersexes were cytogenetically investigated. A sry-negative XX-sex reversal syndrome was diagnosed in a Jack Russel Terrier. In a mixbred dog a persistent Mullerian duct syndrome (PMDS) was found and a Border Terrier Dog showed an XX/XY chromosomal chimaerism. In further four dogs of different breeds, a female constitution of sex chromosomes was seen. As a sign of intersexuality each of these dog showed an enlarged clitoris. A differentiation between XX-sex reversal syndrome and female pseudohermaphroditism was not possible because there was no information on the internal genital tract and gonads available.

Length and somatic mosaicism of CAG and GGN repeats in the androgen receptor gene and the risk of prostate cancer in men with benign prostatic hyperplasia.

BACKGROUND: The most common malignancy in men worldwide is cancer of the prostate and determinants of prostate cancer (PRCa) risk remain largely unidentified. Many candidate genes may be involved in PRCa, such as those that are central to cellular growth and differentiation in the prostate gland. We analysed the polymorphic CAG and GGN repeats sequence in exon 1 of the AR gene to determine if the number of repeats might be an indicator of PRCa risk in patients with BPH. METHODS: The study evaluated 28 patients who presented with PRCa at least 6 years after the diagnosis of BPH and 56 matched patients with BPH who did not progress to PRCa over a comparable period. RESULTS: This study showed no evidence for association between the size of AR CAG and GGN repeats and the risk of the development of PRCa in patients with BPH. However, BPH patients with AR CAG instability had a 12-fold increased risk in development of PRCa. CONCLUSIONS: While independent confirmation is required in further studies, these results provide a potential tool to assist prediction strategies for this important disease.

[Turner's syndrome in mother and daughter]. / Syndroom van Turner bij moeder en dochter.

A girl with Turner's syndrome due to a 45,X mosaicism and a ring chromosome was born to a 29-year-old mother with a non-mosaic 45,X in her blood lymphocytes. Cytogenetic investigation revealed that the ring chromosome of the daughter included almost the entire X chromosome with the exception of the uppermost part of the short arm. In the literature, girls with Turner's syndrome are said to have functional ovarian tissue and pregnancies in women with Turner's syndrome after oocyte donation and intracytoplasmatic sperm injection (ICSI) are no longer exceptional. However, since ovarian failure occurs relatively early during adolescence, cryopreservation of ovarian tissue should be considered as soon as the girl or her parents are able to make the necessary decisions. On the other hand, beside risks for congenital anomalies in the newborn, the risks of pregnancies in Turner's syndrome should not be neglected, notably premature delivery due to disproportion between the pelvis and the foetus and aortic dissection in the pregnant woman.

Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism.

The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.

Malignant refractory epilepsy in identical twins mosaic for a supernumerary ring chromosome 19.

We report identical twins with supernumerary ring chromosome 19 mosaicism, who had severe refractory epilepsy at an early age. The epilepsy was dominated largely by severe life-threatening tonic seizures. Both twins died, likely as a consequence of their severe epilepsy. They displayed no dysmorphic features. Eight cases of ring chromosome 19 have been reported in the literature, all to our knowledge without epilepsy. The clinical picture of these twins emphasizes the importance of carrying out a karyotype study on patients with early-onset epilepsy even in the absence of dysmorphic features.

Ocular manifestations of mosaic trisomy 22: a case report and review of the literature.

Mosaic trisomy 22 is rare, but can be compatible with prolonged life. Patients with mosaic trisomy 22 usually present with intrauterine growth retardation, mental retardation, failure to thrive, and craniofacial asymmetry. We report the case of a five-year-old boy who had a birth weight of 3.8 kg and normal developmental milestones. He presented with unilateral ocular manifestations of ptosis, double elevator palsy, high myopia, and choroidal coloboma involving the macula. Cytogenetic evaluation showed a low level of trisomy 22 in peripheral blood lymphocytes (1 in 100) and in cultured fibroblasts from a conjunctival biopsy of the affected eye (1 in 60). Our case demonstrates the value of chromosomal analysis of the tissues involved rather than just karyotyping of the blood lymphocytes to detect mosaicism in patients with localised and unilateral congenital malformations.

Emergence in Italy of a Neisseria meningitidis clone with decreased susceptibility to penicillin.

A rise in invasive diseases due to Neisseria meningitidis C:2b:P1.5 with decreased penicillin susceptibility occurred in Italy during the last 2 years. Real-time PCR identified the Peni phenotype, and the penA sequence revealed the mosaicism of the gene. Molecular analyses assigned the isolates to a single emergent clone of the hypervirulent A4 cluster.

Screening for mutations in the SRY gene in patients with mixed gonadal dysgenesis or with Turner syndrome and Y mosaicism.

OBJECTIVE: To investigate the presence of mutations in the open reading frame (ORF), as well as on the 5' and 3', flanking regions of the SRY gene in patients with mixed gonadal dysgenesis (MGD) or with Turner syndrome (TS) and Y mosaicism. STUDY DESIGN: We studied 13 patients with MGD and three patients with TS and Y mosaicism. DNA was isolated from blood leukocytes for subsequent polymerase chain reaction (PCR) and direct sequencing were performed in the ORF, as well as from the 5' and 3' flanking regions of the SRY gene. RESULTS: No mutations were present in any of the patients studied. CONCLUSION: The absence of mutations in these regions indicated that mutations were an unlikely cause of MGD or TS with Y mosaicism and suggested that there are others genes playing an important role in sex development.

Somatic mosaicism in FSHD often goes undetected.

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD1A) is associated with contractions of the polymorphic D4Z4 repeat array on chromosome 4qter. The disease has a high frequency of new mutations of mitotic origin. Pulsed-field gel electrophoresis-based studies show that mitotic mutations leading to somatic mosaicism occur equally frequently in patients and parents. Nevertheless, somatic mosaicism in FSHD is mainly reported in asymptomatic parents by applying standard Southern analysis after linear gel electrophoresis. Explaining this apparent discrepancy, we here demonstrate that somatic mosaicism in FSHD patients goes largely undetected using the standard diagnostic technique, indicating that linear electrophoresis is unsuitable to identify mosaic patients. As a consequence, the phenotype of mosaic patient's offspring will be underestimated, whereas the recurrence risk in the symptomatic mosaic individuals will be overestimated. Moreover, somatic mosaicism may partly explain the observation of anticipation in de novo kindreds. Therefore, clinicians should always consider pulsed-field gel electrophoresis analysis in de novo FSHD families, in particular when the patient's phenotype is much milder than expected based on D4Z4 length proper.

Mechanism and timing of mitotic rearrangements in the subtelomeric D4Z4 repeat involved in facioscapulohumeral muscular dystrophy.

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD1A) is associated with contractions of the polymorphic D4Z4 repeat on chromosome 4qter. Almost half of new FSHD mutations occur postfertilization, resulting in somatic mosaicism for D4Z4. Detailed D4Z4 analysis of 11 mosaic individuals with FSHD revealed a mosaic mixture of a contracted FSHD-sized allele and the unchanged ancestral allele in 8 cases, which is suggestive of a mitotic gene conversion without crossover. However, in 3 cases, the D4Z4 rearrangement resulted in two different-sized D4Z4 repeats, indicative of a gene conversion with crossover. In all cases, DNA markers proximal and distal to D4Z4 showed no allelic exchanges, suggesting that all rearrangements were intrachromosomal. We propose that D4Z4 rearrangements occur via a synthesis-dependent strand annealing model that relatively frequently allows for crossovers. Furthermore, the distribution of different cell populations in mosaic patients with FSHD suggests that mosaicism here results from D4Z4 rearrangements occurring during the first few zygotic cell divisions after fertilization.

Quantitative determination of mosaic GFP gene expression in tobacco.

A specific form of gene silencing that was observed visually as a mosaic distribution of fluorescent and non-fluorescent cells apparently dispersed at random within tissues was found in a few green fluorescent protein (GFP)-transformed tobacco lines. To characterize this event quantitatively, we studied flow cytometric measurements in GFP-expressing and -silenced cells in T1 and T2 progeny of four selected plants. The proportion of silenced cells varied considerably among the T1 lines but with notable genotype differences. Mosaic expression was inherited into the T2 generation in which the majority of progenies tested exhibited a level of silencing similar to that of their T1 parental plants. However, in some T2 progenies segregation, evident as a decrease or increase in the proportion of fluorescent cells, was observed. We discuss several factors, such as copy number, promoter activity or polyploidy, that may be the possible causes of the gene silencing, but none sufficiently explain the appearance of the mosaic distribution.

PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves.

Initiation and growth of leaf blades is oriented by an adaxial/abaxial axis aligned with the original axis of polarity in the leaf primordium. To investigate mechanisms regulating this process, we cloned the Nicotiana tabacum ortholog of PHANTASTICA (NTPHAN) and generated a series of antisense transgenics in N. sylvestris. We show that NSPHAN is expressed throughout emerging blade primordia in the wild type and becomes localized to the middle mesophyll in the expanding lamina. Antisense NSPHAN leaves show ectopic expression of NTH20, a class I KNOX gene. Juvenile transgenic leaves have normal adaxial/abaxial polarity and generate leaf blades in the normal position, but the adaxial mesophyll shows disorganized patterns of cell division, delayed maturation of palisade, and ectopic reinitiation of blade primordia along the midrib. Reversal of the phenotype with exogenous gibberellic acid suggests that NSPHAN, acting via KNOX repression, maintains determinacy in the expanding lamina and sustains the patterns of cell proliferation critical to palisade development.

Chromosomal abnormalities in fetuses with open neural tube defects: prenatal identification with ultrasound.

OBJECTIVES: To determine the prevalence of chromosomal abnormalities in fetuses with open neural tube defects (NTD) undergoing prenatal chromosome analysis. The role of prenatal ultrasound in detecting those with an underlying chromosomal abnormality was also investigated. METHODS: Over a 6-year period, 144 fetuses with open NTD underwent prenatal chromosome analysis between 12 and 37 weeks of gestation, as part of a prospective, multicenter prenatal diagnosis and counseling program in Chile. This population included 66 fetuses with spina bifida, 46 with acrania/anencephaly, 21 with cephalocele and 11 with iniencephaly. A confident prenatal diagnosis was made in 143 fetuses (99%) and confirmed postnatally in all cases. RESULTS: An underlying chromosomal abnormality was diagnosed in 10 fetuses (7%), six with spina bifida, three with cephalocele and one with craniorachischisis. The prevalence of chromosomal abnormality varied according to the defect present in the fetus, with a 14% (3/21) prevalence among those with cephalocele, 9% (6/66) among those with spina bifida and 2% (1/57) among those with lethal defects such as acrania, anencephaly or iniencephaly. Karyotype results revealed trisomy 18 in seven cases, trisomy 13 in two and mosaicism for a marker chromosome in one. Prenatal ultrasound before the procedure showed that all chromosomally abnormal fetuses had additional findings. The prevalence of chromosomal abnormality in fetuses with spina bifida and cephalocele was higher when chromosome analysis was performed at or before 24 weeks of gestation in comparison to those performed after 24 weeks (5/31 (16%) vs. 4/56 (7%), respectively). However, this difference did not reach statistical significance, probably due to the small number of cases. CONCLUSIONS: A significant number of fetuses with open NTD are chromosomally abnormal. Although prenatal chromosome analysis should be considered in all cases, prenatal ultrasound seems effective in identifying those fetuses with an underlying chromosomal abnormality.

Arg120stop nonsense mutation in the RP2 gene: mutational hotspot and germ line mosaicism?

Mutations in the RP2 gene account for up to 20% of X-linked recessive retinitis pigmentosa (RP). Arg120stop is to date the most frequently reported mutation found in RP2. Mutation screening was performed during the course of a large screening program of retinal degenerative disorders (RDDs) in South Africa using exon 1 and 2 of RP2 in 20 unrelated families with an X-linked mode of retinal degenerative inheritance. Direct sequencing analysis revealed a C-->T transition at position 358 in the proband in a family of German origin. Subsequent analysis revealed that this Arg120stop mutation cosegregated with the disease in an additional affected family member. The nonsense mutation, Arg120stop, could not however, be detected in the somatic cells of the obligate carrier female. This, the first report of a germ line mutation for a family with RP, has many implications for genetic counseling of retinal degeneration (RD). To avoid inaccurate risk assessment for RP due to epigenetic events, such as the rare occurrence of germ line mosaicism, genetic counseling in families with XLRP should always be guided by molecular testing.

Growth of heterokaryotic monozygotic twins discordant for Ullrich-Turner syndrome during the first years of life.

The rare observation of different karyotypes in monozygotic (MZ) twins, i.e., heterokaryotic monozygosity, occurs due to chromosomal aberration in one of the twins after separation of the embryos. We report on the differences of heterokaryotic MZ Turkish twins who are discordant for Ullrich-Turner syndrome. Chromosomal analyses from peripheral lymphocytes revealed a 45,X/46,XX mosaicism in both twins. FISH analyses of buccal smears showed 99% of nuclei 45,X in twin A and 98% of nuclei 46,XX in twin B. These results are consistent with a non-mosaic 45,X and 46,XX karyotype, respectively. The girls showed a different growth pattern in the first years. As their genotype should be identical except for the number of X chromosomes, the difference in phenotype may be a pure result of loss of one X chromosome in the affected girl. Special interest is set on the spontaneous and growth hormone induced growth of the twins.

High proportion of mutant osteoblasts is compatible with normal skeletal function in mosaic carriers of osteogenesis imperfecta.

Individuals with mosaicism for the autosomal dominant bone dysplasia osteogenesis imperfecta (OI) are generally identified by having more than one affected child. The mosaic carriers have both normal and mutant cell populations in somatic and germline tissues but are unaffected or minimally affected by the type I collagen mutation that manifests clinically in their heterozygous offspring. We determined the proportion of mutant osteoblasts in skeletal tissue of two mosaic carriers who each have a COL1A1 mutation in a high proportion of dermal fibroblasts. Both carriers had normal height and bone histology; the first carrier had normal lumbar spine measurements (L1-L4), as determined by dual-energy x-ray absorptiometry (Z = +1.17). In cultured cells from the first carrier, studied by labeled PCR and single-cell PCR over successive passages, the collagen mutation was present in 85% of fibroblasts and 50% and 75% of osteoblasts from her right iliac crest and left patella, respectively, with minimal selection. The second carrier was studied by PCR amplification of DNA from autopsy paraffin blocks. The proportion of heterozygous cells was 40% in calvarium, 65% in tracheal ring, and 70% in aorta. Thus, in OI, substantially normal skeletal growth, density, and histology are compatible with a 40%-75% burden of osteoblasts heterozygous for a COL1A1 mutation. These data are encouraging for mesenchymal stem-cell transplantation, since mosaic carriers are a naturally occurring model for cell therapy.