Genetic Imbalance Research Articles

545 – Paranoid schizophrenia in kallmann syndrome: genetics and psychopathology

Introduction Kallmann syndrome (KS) is a genetically heterogenous and rare disorder characterized by the combination of hypothalamic hypogonadism and anosmia/hyposmia. In about one third of the patients, mutations have been identified in at least seven different genes. Apart from one published case report, virtually no data are available about possible neuropsychiatric symptoms in KS. Objectives Studying the putative relationship between KS and schizophrenia. Aims Investigating the genetic etiology in a patient with a clinical diagnosis of KS and paranoid schizophrenia. Methods Detailed diagnostic evaluation including genetic analysis of an adult male patient with paranoid schizophrenia, hypogonadism and anosmia. Results Psychiatric examination of the 28-years-old male patient disclosed negative psychotic symptoms only, following an acute paranoid schizophrenic episode one year before. He used 15mg aripiprazole daily and testosterone gel 20mg/gr. Hormonal panel showed extremely low levels of LH and FSH. MRI scanning of the brain demonstrated complete absence of the olfactory bulbs. Otolaryngeal examination disclosed complete anosmia. Neuropsychological assessment revealed subaverage intelligence distinctly disconcordant with premorbid functioning, markedly lowered speed of information processing, and impaired executive and social cognitive functions. Genome wide SNP array analysis and mutation analyses for the KAL1, FGFR1, PROK2, PROKR2, FGF8 and CHD7 genes, disclosed no genetic imbalance or any pathogenic mutation. Conclusions This first report on a patient with KS and paranoid schizophrenia in whom extensive genetic analyses were performed, stipulates the need for studying a possible increased risk for psychiatric symptoms in patients with KS.

A novel maternally‐derived insertional translocation resulting in partial trisomy 4q13.2–q22.1 with complex translocation t(8;20) in a family with intellectual disability

We characterized the chromosomal aberration in family with intellectual disability, including two affected children and their affected mother. Initial standard karyotypes of the three individuals showed an apparently balanced translocation of chromosomes 8 and 20. Using molecular cytogenetic techniques, we observed complex structural chromosomal aberration comprising of reciprocal translocation between chromosomes 8 and 20 with pericentric inversion (8p11.12q22.3) and insertion of chromosome 4 segments into both der(8) and der(20). In particular, the insertion of chromosome 4 was complex. Two segments (4q13.2-q13.3 and 4q21.21-q22.1) were inserted into the der(8)t(8;20) breakpoint and one segment (4q13.3-q21.21) into the der(20)t(8;20) breakpoint. Both children inherited two normal chromosomes 4 from their parents and the der(8) and der(20) from the mother, resulting in partial trisomy of 4q13.2-q22.1. Interestingly, the mother, in addition to the same complex insertions and inversion, was founded to have a deletion of 4q13.2-q22.1 in one of her chromosomes 4, yielding no genetic imbalance but with potential disruption of intellectual dysfunction-related gene(s) at the breakpoints as the cause of her intellectual impairment. This family is the third case report of an insertional translocation mechanism causing partial trisomy 4q syndrome. Our study demonstrates that an insertion of an extra chromosomal segment, not primarily involving in translocation breakpoints, which results in partial trisomy, can be an unapparent cause of the abnormal phenotypes.

Association of KIR3DS1+HLA-B Bw4Ile80 combination with susceptibility to tuberculosis in Lur population of Iran.

Natural killer (NK) cells are the effector cells of innate immunity that respond to infection and tumor. Interactions between killer cell immunoglobulin like receptors (KIR) and human leukocyte antigen (HLA) class I molecules regulate NK cells responses to eliminate infected and transformed cells. To investigate the impact of KIR genes, HLA ligand genes, and KIR-HLA combinations on susceptibility to tuberculosis (TB) in Lur population of Iran. The genomic DNA of 50 patients with TB from Lorestan province of Iran was genotyped for sixteen KIR genes and their five major HLA class I ligands were determined by a polymerase chain reaction-sequence-specific primers (PCR-SSP) assay. The results were compared with those of 200 healthy unrelated Iranian individuals. In Lur population of Iran, a significant decrease in frequency of KIR3DS1 was found in TB patients compared to control group (24% vs. 44.5%, OR=0.394, CI=0.194-0.798, p=0.013). Also, among the three activating genes that may use HLA class I molecules as their ligands, a significant decrease was shown in frequency of KIR3DS1 with HLA-B Bw4Ile80 ligand in TB patients compared to control group (4% vs. 23%, OR=0.14, CI=0.033-0.596, p=0.004). These findings imply a genetic imbalance between activating and inhibitory KIR genes and KIR-HLA combinations in Lur TB patients. Low level of activating KIR3DS1 and its combination with HLA-B Bw4Ile80 ligand might have an influence on the susceptibility to TB in Lur population of Iran.

Spontaneous Transformation of Stem Cells In Vitro and the Issue of Cross-Contamination

We have read with interest the paper “Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells”, recently published by Wu et al. 1. In this study the authors show spontaneous transformation of human fetal striatum neural stem cells (hsNSCs) in culture, and that the transformed cells (T1) are characterized by stem cell-like features, the expression of neural stem cell markers, abnormal karyotype and an increased growth rate. In the text they refer to previous reports on spontaneous MSC transformation 2, 3. However, they fail to inform the readers that both these publications have later been retracted or corrected since both research groups detected that their transformed cells were cross-contaminated with cancer cells 4, 5. In the article by Wu and colleagues, they have characterized the T1 cells by DNA fingerprinting. Most interesting, the DNA fingerprint of the transformed cells (T1) did not match the “cell of origin”, and the authors explain this by genetic instability. However, we have compared the T1 fingerprint published by Wu et al., with public available cell line STR profiles, and find that the T1 STR profile published by Wu et al. is surprisingly similar to HeLa cells, Table ​Table1.1. The DNA fingerprinting profile of cancer cells compared to normal cells is characterized by large differences in peak height at one or more loci, indicating genetic instability, occasional additional alleles at a locus, indicating gene duplication events, and loss of heterozygosity (LOH), at one or more loci 6, 7. Genetic imbalance will in other words not generate a completely new fingerprinting profile. Table 1 STR fingerprinting profile of hsNSC, T1 and HeLa STR profiling is currently the recommended test for cell line authentication due to its high power of discrimination and the possibility to compare the numerical code obtained from various laboratories 7. Wu and colleagues analyzed their cells by using the PowerPlex 16 System Kit from Promega. The kit provides 15 STR markers as well as the gender determinator Amelogenin, and it has a matching probability of > 1 in 1.83×10e17 (www.promega.com). The same kit was recently used to determine the STR profile of HeLa cells 8, showing 97% identity between T1 and HeLa with only one LOH (Table ​(Table1).1). According to general recommendations, the profile of identical or closely related profiles should match at 80% or more of the alleles 6, and profiles with an identity level between 50 and 75% must be regarded with suspicion 7. The HeLa profiles listed in Table ​Table11 match T1 with 80-97% accuracy. A minor variation is seen at one locus when comparing the STR profile reported by ATCC and CLS (D13S317: 12,13.3 and 13,13.3) and Wu (D13S317: 12,14). According to the Promega Protocol for PowerPlex16, each allele at the D13S317 locus separates by 4 nucleotides, and it is therefore unclear if the allele at D13S317 13.3 is correct. There is at present several batches of HeLa cells available, and minor differences exists between them 6. A number of scientists have pointed at the problem of cross-contamination for decades, and it is now highly recommended to authenticate cells by DNA fingerprinting 7, 9. Several databases for checking the fingerprinted profiles are available, such as STR profile databases at ATCC (www.lgcstandards-atcc.org) and DSMZ (www2.dsmz.de). Also a list of 360 cross-contaminated cell lines is available to help researchers quality-check their work 10, and HeLa is still the most frequent cross-contaminating cell line 10. In conclusion, it is highly questionable if the article presented by Wu et al., actually describes a transforming event of hsNSCs.

Genomic differences between estrogen receptor (ER)‐positive and ER‐negative human breast carcinoma identified by single nucleotide polymorphism array comparative genome hybridization analysis

Estrogen receptor (ER) remains one of the most important biomarkers for breast cancer subtyping and prognosis, and comparative genome hybridization has greatly contributed to the understanding of global genetic imbalance. The authors used single-nucleotide polymorphism (SNP) arrays to compare overall copy number aberrations (CNAs) as well as loss of heterozygosity (LOH) of the entire human genome in ER-positive and ER-negative breast carcinomas. DNA was extracted from frozen tumor sections of 21 breast carcinoma specimens and analyzed with a proprietary 50K XbaI SNP array. Copy number and LOH probability values were derived for each sample. Data were analyzed using bioinformatics and computational software, and permutation tests were used to estimate the significance of these values. There was a global increase in CNAs and LOH in ER-negative relative to ER-positive cancers. Gain of the long arm of chromosome 1 (1q) and 8q were the most obvious changes common in both subtypes: An increase in the chromosome 1 short arm (1p)/1q ratio was observed in ER-negative samples, and an increased 16p/16q ratio was observed in ER-positive samples. Significant CNAs (adjusted P<.05) in ER-negative relative to ER-positive tumors included 5q deletion, loss of 15q, and gain of 2p and 21q. Copy-neutral LOH (cnLOH) common to both ER-positive and ER-negative samples included 9p21, the p16 tumor suppressor locus, and 4q13, the RCHY1 (ring finger and CHY zinc finger domain-containing 1) oncogene locus. Of particular interest was an enrichment of 17q LOH among the ER-negative tumors, potentially suggesting breast cancer 1 gene (BRCA1) mutations. SNP array detected both genetic imbalances and cnLOH and was capable of discriminating ER-negative breast cancer from ER-positive breast cancer.

Microarray Analysis in Children With Developmental Disorder or Epilepsy

The technique of chromosomal microarray analysis identifies genetic imbalance. Evaluation of its diagnostic role in pediatrics is still underway. We describe our experience with chromosomal microarrays. We retrospectively reviewed the charts of children in the Sections of Neurology and Clinical Genetics at St. Christopher's Hospital for Children who had undergone microarray analysis between 2006 and 2009. Collected data included age, sex, and the presence of mental retardation, developmental delay, autism, learning disability, hypotonia, dysmorphic features, and epilepsy, and the use of microarray technique. Statistical analysis was performed using SPSS. There were 82 children (mean age ± S.D., 5.7 ± 5 years), including 45 (55%) boys and 37 (45%) girls. All patients exhibited a normal karyotype. Microarray analysis produced abnormal results in 20 (23.5%). Deletions comprised 74% of all abnormalities. Patients with ≥ 4 clinical variables demonstrated a 30.5% incidence of abnormal chromosomal microarray findings, compared with 8.7% of patients with ≤ 3 clinical variables (P = 0.039, χ(2) test). Logistic regression indicated that motor impairment (P = 0.039) and presence of epilepsy (P = 0.024) independently contributed to the model. The likelihood of an abnormal microarray result increased with the number of clinical abnormalities. Microarray analysis will likely become the diagnostic genetic test of choice in children with neurodevelopmental disorders or epilepsy.

Allelic imbalance and abnormal expression of FHIT in endemic nasopharyngeal carcinoma: association with clinicopathological features

The FHIT gene is involved in the pathogenesis of many cancers. The aim of this study was to investigate allelic imbalance (AI) pattern at FHIT locus and alteration of FHIT gene in nasopharyngeal carcinoma (NPC) and analyzed potential correlation between AI, FHIT mRNA expression and clinicopathological factors. We examined AI, including loss of heterozygosity (LOH) and microsatellite instability (MSI), at FHIT locus in 41 cases of NPC by microsatellite analysis and FHIT gene status in 30 cases of NPC by nested reverse transcriptase-polymerase chain reaction and DNA sequencing. The frequencies of LOH and MSI at FHIT locus in NPC were 70.7% (29/41) and 36.6% (15/41), respectively. Thirteen of thirty (43.3%) NPCs exhibited aberrant FHIT transcripts. LOH and abnormal FHIT expression were correlated with advanced clinical stage and higher titers of immunoglobulin (Ig) A against Epstein-Barr virus capsid antigen (EBVCA-IgA) (p < 0.05). Abnormal FHIT expression was also correlated with tumor recurrence (p < 0.05). MSI was correlated with early clinical stage and higher titers of EBVCA-IgA (p < 0.05). AI at FHIT locus is a common event and contributes to genetic imbalance in NPC. The abnormalities of FHIT, presumably associated with genetic imbalance at FHIT locus, might be involved in the development and the tumor recurrence of NPC.

Correlation analysis of CYP2B6 gene polymorphisms and pharmacokinetic parameters of isotretinoin in healthy human volunteers.

Objective To evaluate the association between isotretinoin pharmacokinetic parameters and CYP2B6 (cytochrome p-450) gene polymorphisms. Methods Blood samples were collected at different time points from 21 healthy male volunteers who received a single 40-rng oral dose of isotretinoin. High performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS) was used for the quantification of isotretinoin in plasma samples which were standardized by dosage and body weight. PCR and restriction fragment length polymorphism (RFLP) analysis were performed to detect the G516T mutation in exon 4 as well as A785G mutation in exon 5 of CYP2B6 gene in these subjects. Results There was an obvious genetic linkage imbalance in exon 4 and 5 of CYP2B6 gene among these volunteers. In the case of CYP2B6*4 allele,3 (14.29%) people were CYP2B6*4/*4 homozygotes, 6 (28.57%) CYP2B6*1/*4 heterozygotes, and 12 (57.14%) CYP2B6*1/*1 wild-type homogygotes, while as far as CYP2B6*6 allele was concerned, 3 (14.29%)people were CYP2B6*6/*6 homozygotes, 5 (23.81%) CYP2B6*1/*6 heterozygotes, and 13 (61.90%)CYP2B6*1/*1 wild-type homozygotes. The reaction half-time (t1/2) and mean residence time (MRT) of isotretinoin were longer in volunteers carrying wild-type CYP2B6*4 allele than those of CYP2B6*4/*4 homozygotes (both P ＜ 0.05 ), while no significant difference was observed in maximum concentration (Cmax), peak time (Tmax) or area under the plasma concentration time (AUC) between the two groups of volunteers. There was no statistical difference in any of the above parameters between subjects carrying wild type CYP2B6*6 allele and those of CYP2B6 *6/*6 homozygotes (all P ＞ 0.05 ). Conclusions The mutation of CYP2B6*4 allele ia relevant to the metabolism of isotretinoin, which seems to be more rapid in CYP2B6*4/*4 homozygotes. Key words: Isotretinoin; Pharmacokinetics; Cytochrome P-450, CYP2B6

Genetic copy number variants in sib pairs both affected with schizophrenia.

BackgroundSchizophrenia is a complex disorder with involvement of multiple genes.MethodsIn this study, genome-wide screening for DNA copy-number variations (CNVs) was conducted for ten pairs, a total of 20 cases, of affected siblings using oligonucleotide array-based CGH.ResultsWe found negative symptoms were significantly more severe (p < 0.05) in the subgroup that harbored more genetic imbalance (n ≧ 13, n = number of CNV-disrupted genes) as compared with the subgroup with fewer CNVs (n ≦ 6), indicating that the degree of genetic imbalance may influence the severity of the negative symptoms of schizophrenia. Four central nervous system (CNS) related genes including CCAAT/enhancer binding protein, delta (CEBPD, 8q11.21), retinoid × receptor, alpha (RXRA, 9q34.2), LIM homeobox protein 5 (LHX5, 12q24.13) and serine/threonine kinase 11 (STK11, 19p13.3) are recurrently (incidence ≧ 16.7%) disrupted by CNVs. Two genes, PVR (poliovirus receptor) and BU678720, are concordantly deleted in one and two, respectively, pairs of co-affected siblings. However, we did not find a significant association of this BU678720 deletion and schizophrenia in a large case-control sample.ConclusionsWe conclude that the high genetic loading of CNVs may be the underlying cause of negative symptoms of schizophrenia, and the CNS-related genes revealed by this study warrant further investigation.

Variation in pollen formation and its cytological mechanism in an allotriploid white poplar

Variation in pollen formation and its cytological mechanism in an allotriploid white poplar were investigated by the squashed technique and indirect immunofluorescence. Besides 0.5% stuck pollen grains, this allotriploid produced regularly spherical pollen grains. It was estimated that 90.3% of pollen grains were viable. Diameters of the viable spherical pollen grains ranged from 23.2 to 72.9 μm, with a bimodal frequency distribution. Numerous meiotic abnormalities were found, including highly irregular chromosome pairing, lagging chromosomes and chromosome bridges, micronuclei, and multiple spindles, which indicate highly genetic imbalance of this allotriploid. Some micronuclei triggered minispindle formation in metaphase II and participated in cytokinesis to form microcytes in sporads. Abnormal orientation of metaphase II spindles contributed to production of dyads and triads, which produced unreduced microspores. However, parallel orientation of spindles was not necessary for dyad formation, because an organelle band positioned in the equatorial region prevented the spindles from coalescing. Some microsporocytes exhibited a complete or partial absence of cytokinesis, which resulted in the formation of monads and the increased frequency of dyads and triads. The perspective of this triploid in the polyploid breeding program of white poplar is discussed.

Foreword: Coping with sex chromosome imbalance

This special issue is devoted to the topic of dosage compensation. The dramatic phenotypes that are associated with most autosomal monosomies or trisomies illustrate the importance of maintaining appropriate gene dosage in diploid organisms. Buffering and compensating for such genetic imbalance clearly represents a challenge for complex eukaryotes. In sexually dimorphic species with chromosome-based mechanisms of sex determination (XX/XY and ZW/ZZ), the contrasting doses of sex chromosomes in males and females would be expected to introduce a large-scale imbalance in levels of gene expression. This imbalance would be at two levels. First, the dosage differences between the sexes (XX versus XY or ZW versus ZZ); second, the dosage difference between sex chromosomes and autosomes in the heterogametic sex (XY males, or ZW females) where only a single copy of the X or Z chromosome is present, in contrast to two copies of all autosomes. However, sex chromosome imbalances are usually well tolerated—how can this be?

The in vivo modulatory effects of an anterior-chamber microenvironment on uveal melanoma

Background:Primary melanoma of the iris, for reasons unknown has a lower metastatic rate compared with primary ciliary-body melanoma. Six histology cases of ciliary-body melanoma were identified that had spread onto...

Common Submicroscopic Genomic Imbalances Accompany the Ph Chromosome at Diagnosis in Chronic Myeloid Leukemia

Despite the presence of a consistent genetic abnormality, CML patients display considerable clinical heterogeneity, the basis of which is poorly understood. We therefore used a novel ultra-high-resolution genomic screening assay to search for additional acquired genomic abnormalities that might explain this clinical heterogeneity and help to assess prognosis for individual patients. Comparative genomic hybridisation (CGH) was performed with a 2.1 million oligonucleotide array on DNA extracted from presentation samples of 20 previously untreated CML patients. Each DNA sample from diagnosis was competitively hybridized against the same patient's remission sample, which avoided detection of constitutional polymorphic copy number variants (CNVs). For representative copy number aberrations (CNAs) the CGH result was confirmed by fluorescence in situ hybridization or quantitative real-time PCR. All 20 CML patient samples harbored detectable genomic imbalances with a median of 16 CNAs per patient (range: 4–165). Of the 846 CNAs detected 679 (80%) were amplifications and 167 (20%) were deletions. 462 CNAs (55%) involved at least one known gene. The median size of CNAs was 58kb (range 9kb – 12Mb). One hundred and twenty seven different genomic regions were aberrant in 2 or more patients in the cohort. Of these recurrent CNAs, amplifications of the FOXD4L4, MAPK8IP1, DUSP1, PSTPIP1 and PBEF1 genes were among the most frequently detected; they were present in 3, 4, 5, 6 and 15 patients respectively. FOXD4L4 is a myeloid trancription factor previously implicated in leukemogenesis, and PSTPIP1 has been shown to be involved in dephosphorylation of ABL. MAPK81P1 and DUSP1 are both involved in the MAPK pathway, while PBEF1 is known to play a role in the NAD anti-apoptotic pathway. The presence of multiple genomic lesions at diagnosis supports the notion of an increased level of genomic instability in CML cells and raises the possibility that one or more aberrations in addition to BCR-ABL may dictate the CML phenotype. The considerable range in the number of CNAs identified at diagnosis may reflect a differing level of genomic instability in different patients. Furthermore the presence of same genetic imbalance in more than one patient suggests a role for specific genes in addition to BCR-ABL in the pathogenesis or progression of CML.

Down syndrome and the enteric nervous system

Down syndrome (DS) is the most common chromosomal abnormality occurring in humans. Up to 77% of DS children have associated gastrointestinal (GI) abnormalities, which may be structural or functional in nature. Functional disturbances may, in turn, affect the outcome of corrective surgical procedures, prompting to caution. It is becoming clear that the processes affecting the enteric nervous system (ENS) in DS not only affect the micro-anatomy but also nerve function, and there is some histological evidence of ENS variations in both human and DS animal models. This suggests that developmental disorders of the ENS are probably fundamental to the functional GI disturbances encountered in patients with DS. The anomalous brain development, function and resulting intellectual impairment associated with DS appears to result from the genetic imbalance created by the trisomy of chromosome 21. The possible links between the brain, GI and ENS involvement are not as yet entirely clear. Neurotropic factors affecting brain development during embryogenesis are probably interlinked with ENS development, but the precise mechanism of how this occurs has yet to be established. This study explores what is known about the ENS dysfunction in DS and reviews the possible importance of chromosome 21 located and other genes in its etiology. Functional motor disturbances of the esophagus and colon are not uncommon and may be congenital or acquired in nature. The most prominent of these include esophageal dysmotility syndromes (e.g. achalasia, gastroesophageal reflux, dysphagia) as well as a higher incidence of chronic constipation and Hirschsprung's disease (HSCR) (2-15%) occurring in association with DS. Chromosome 21 itself is thought to be the site of a modifier gene for HSCR. Recently identified candidate genetic mechanisms provide unique insights into the genetic background of the neurological and cognitive disorders associated with DS. Although the role of the triplicated chromosome 21 and genetic dosage remain important, the additional role of other chromosome 21 genes in the etiology of ENS developmental anomalies remains undetermined and requires ongoing research.

Clinical and pathological aspects of thymic epithelial tumors

A histological classification of thymic epithelial tumors was presented by the World Health Organization (WHO) in 1999 and again in 2004 following slight modifications, in which thymic epithelial tumors were categorized as thymomas and thymic carcinomas. Whereas thymoma is defined as an organotypic (thymus-like) tumor, thymic carcinoma is a malignant epithelial neoplasm with a morphology similar to that of malignant neoplasms arising from other organs. Herein, the recent progress in research of thymic epithelial tumors is reviewed with reference to the WHO histological classification system, with the focus on thymomas. Thymomas are classified into five types--A, AB, B1, B2, B3--according to the shape and atypia of their epithelial cells as well as the abundance of lymphocytes. The invasiveness, prognosis, and genetic imbalance of thymomas have been shown to be related to this classification system. Myasthenia gravis is frequently associated with types B1 and B2. The WHO histological classification of thymomas is not only useful for treatment but reflects their biological characteristics, including genetic alterations. Advances are expected in future studies of thymomas from the standpoint of their clinical, pathological, and biological aspects.

Beckwith–Wiedemann‐like macroglossia and 18q23 haploinsufficiency

Beckwith-Wiedemann syndrome (BWS) is an overgrowth condition with tumor proclivity linked to a genetic imbalance of a complex imprinted region in 11p15.5. A female child with features fitting in with the BWS diagnostic framework and an apparent loss of imprinting (LOI) of the IGF2 gene in 11p15.5 was also reported to have a de novo chromosome 18q segmental deletion (Patient 1), thus pointing at the location of a possible trans-activating regulator element for maintenance of IGF2 imprinting and providing one of the few examples of locus heterogeneity of BWS. A second child with de novo 18q23 deletion and features of macroglossia, naevus flammeus, bilateral inguinal hernia and transient neonatal hypoglycemia, thus also fitting in with the BWS diagnostic framework, is here fully reported (Patient 2). In this child, an analysis of the BWS1 locus precluded any paternal isodisomy and showed a normal imprinting pattern (mono-allelic expression of IGF2 and normal H19 and CDKN1OT1/LIT1 methylation index). In Patients 1 and 2, deletions were shown to overlap, defining a minimal region of haplo-insufficiency of 3.8-5.6 Mb in 18q23. We conclude that this region provides a candidate location for an original macroglossia condition with strong overlap with BWS, but without obvious upstream functional relationship with the BWS1 locus in 11p15.5. Because this minimal region of haplo-insufficiency falls into a common region of deletion in 18q- syndrome, we inferred that this macroglossia condition would follow a recessive pattern of inheritance.

Reply from M. Dutschmann and G. M. Stettner

We appreciate the interest and comments regarding our recently published article in The Journal of Physiology (Stettner et al. 2007a). The manuscript described disturbances in vagal-pontine interactions in the control of the postinspiratory activity as a potential cause of breathing disturbances in MECP2-deficient mice, an animal model for Rett syndrome (Guy et al. 2001). The Letter by Poon & Song addresses an aspect of the publication that is illustrated in Fig. 7, which contrasts the response to repetitive electrical stimulation of the vagal nerve of wild-type (C57BL/6J) and MECP2-deficient mice. In both groups of mice, the intensity of electrical stimulation of the central end of the vagus nerve was determined by the same procedure and the stimuli elicited a postinspiratory reflex apnoea (Hering–Breuer reflex). In particular, the intensity of vagal stimulation in our study was 1–2 × above threshold (low intensity stimulation), which was similar to that used to study plasticity following repetitive vagal stimulation (Siniaia et al. 2000). The data showed that the apnoea after cessation of the electrical stimulus was significantly longer in MECP2-deficient mice than in C57BL/6J mice. Furthermore, the reflex apnoea progressively decreased with repeated vagal stimuli in the wild-type but not MECP2-deficient mice. We interpreted our data as Mecp2−/y knockout mice lacked ‘desensitization’ to vagal stimulation. This terminology was criticised in the letter to the Editor. We want to emphasize that disturbed processing of vagal afferent input remains a part of the phenotype of the MECP2-deficient mice, whether they lack desensitization or ‘classic habituation’. We agree that a full description of the underlying plasticity phenomenon would require analysis of the poststimulus rebound activity. At this point, we cannot confirm the correspondent's insights regarding Fig. 7. Differences in the stimulus intensities between our and other studies relate to different experimental tools, for instance the central vagal nerve was stimulated via suction electrodes in our experiment. This may clarify confusion regarding a potential secondary or non-specific sensitization of the vagally evoked reflex response due to activation of rapidly adapting pulmonary stretch receptor afferent fibres or cardiopulmonary C-fibres. Even though group data for the poststimulus rebound were not analysed, Fig. 7 shows that expiration lengthened after the stimulus. Poststimulus rebound activity as described for rats (Siniaia et al. 2000), however, did not occur in C57BL/6J and MECP2-deficient mice. The C57BL/6J mouse strain itself may have an imbalance in ponto-medullary circuits. We have observed spontaneous postinspiratory apnoeas in the perfused brainstem preparation in situ (Stettner et al. 2007b) and others report severe periodic breathing patterns during the recovery from a hypoxic challenge in vivo (Han et al. 2002; Gonsenhauser et al. 2004). We speculate that the pontine Kölliker–Fuse nucleus (KF) may be a part of this imbalance, not only because neurones in the KF express postinspiratory activity in many species (Dick et al. 1994; Ezure, 2006; Dutschmann & Herbert, 2006), but also because KF neuronal activity determines the timing of postinspiratory activities of laryngeal muscles in the absence of vagal afferent feedback (Dutschmann & Herbert, 2006). If pontine mechanisms in the control of breathing are similar in rats and mice, then spontaneous postinspiratory apnoeas in C57BL/6J mice indicate a genetic imbalance in pontine mechanisms controlling postinspiratory activity in C57BL/6J mice that may be accentuated under the condition of MECP2 deficiency. This may cause the unusual poststimulus rebound illustrated in Fig. 7. Interestingly, another Mecp2−/y knockout model (Chen et al. 2001) has a different genetic background. If we are correct about the intrinsic imbalance prevalent in C57BL/6J mice, then these other MECP2-deficient mice should show distinct differences in their breathing phenotype. In our opinion, further experiments are required in MECP2-deficient mice with different genetic backgrounds to fully understand disturbed synaptic plasticity associated with the processing of afferent vagal feedback.

Hyperbaric oxygen therapy of angiopathic changes in patients with inherited gene imbalance

Phenotype match inherited by genes is in most cases present in monozygotic twins. Their phenotypic resemblance is unfortunately characterized by strong susceptibility for the development of chronic non-infectious diseases. One of the most common non-infectious chronic diseases that are phenotipically represented in twins is diabetes mellitus. Genetic imbalance is, in most cases, placed in 2, 3, 7, 8, 11, 12, 19 and 20 chromosomal pair of the human genome. This study describes a pair of monozygotic twins, aged 54, who were diagnosed for diabetes type 2 ten years earlier. The first patient had trophic changes of muscles and skin tissues of the lower limb, and a necrotic wound on his right leg tibial region with the claudication distance of 50 m. After arteriography, he was referred by a vascular surgeon for hyperbaric oxygen therapy (HBO). HBO protocol implied 70 min. application of 100% oxygen at 2.5 absolute atmospheres. After the first series of HBO therapies consisting of 20 HBO treatments, claudication was eliminated and the necrotic wound healed. Next, surgical aortofemoral bypass was done. During the second HBO treatment, his monozygotic twin brother presented with angiopathic changes due to diabetes. In both patients, biochemical parameters corresponded to the expected level for diabetes type 2 imbalance, and the localization of the chromosomal defect (placed on 3, 11 and 19 chromosomal pair) was also in accordance with the respective disorder. After they were included into next 10 HBO treatments, Doppler imaging of the major arteries of limbs revealed normal findings. Identical genetic impairment in monozygotic twins can lead to identical somatic changes with resultant consequences. HBO treatment of such patients associated with other therapeutic procedures (conducted by diabetologist, vascular surgeon and physiatrist) can postpone or prevent irreversible changes occurring due to blood vessel disorders.

Xist and the order of silencing

X inactivation is the mechanism by which mammals adjust the genetic imbalance that arises from the different numbers of gene-rich X-chromosomes between the sexes. The dosage difference between XX females and XY males is functionally equalized by silencing one of the two X chromosomes in females. This dosage-compensation mechanism seems to have arisen concurrently with early mammalian evolution and is based on the long functional Xist RNA, which is unique to placental mammals. It is likely that previously existing mechanisms for other cellular functions have been recruited and adapted for the evolution of X inactivation. Here, we critically review our understanding of dosage compensation in placental mammals and place these findings in the context of other cellular processes that intersect with mammalian dosage compensation.

A new genomic mechanism leading to cri‐du‐chat syndrome

Using standard banding techniques, a within-arm intrachromosomal insertion can be mistakenly interpreted as a paracentric inversion. The need to correctly distinguish between these two types of chromosome rearrangements is emphasized by their different reproductive risks. For carriers of an intrachromosomal insertion, the empiric risk of having a liveborn child with a recombinant chromosome leading to a genetic imbalance is at least 15%, whereas the risk for a carrier of a paracentric inversion having a liveborn child with a recombinant chromosome leading to a genetic imbalance is thought to be practically negligible. We report a unique observation in which a paracentric inversion in the short arm of chromosome 5, 46,XX,inv(5)(p13.3p15.3), was identified in a women who had a daughter with an apparently terminal deletion in the distal short arm of chromosome 5, 46,XX,del(5)(p14.3), and the clinical diagnosis of cri-du-chat syndrome. We further characterized the rearrangement, and fluorescence in situ hybridization (FISH) and microsatellite analyses confirmed the paracentric inversion in the mother and showed the deletion in the daughter was maternal in origin. Therefore, this represents a case in which a confirmed paracentric inversion likely resulted in a viable terminal deletion. We propose a mechanism involving dicentric chromosome formation with subsequent breakage and telomere healing during meiosis. This illustrates a new genomic mechanism of chromosome rearrangement leading to cri-du-chat syndrome and should provide significant information for the medical management of patients with other terminal deletion syndromes.