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.

[X-linked recessive ichthyosis (XRI), cerebellar ataxia and neuropsychiatric symptoms]. / X-chromosomal-rezessive Ichthyose (XRI) mit zerebellärer Ataxie, Angsterkrankung und Depression.

X-Linked ichthyosis (XRI) is a keratinisation disorder caused by a mutation of the steroid sulfatase gene. An association with mental retardation and epilepsy has been reported earlier. Here, we report on a patient suffering from cerebellar symptoms such as yes/yes head tremor, scanning dysarthria, pronounced dysmetria and intention tremor, without any abnormalities of the cerebellum in MRI, in addition to XRI proven by molecular genetics. Furthermore, the patient suffered from anxiety disorder, depression, and a male pattern baldness. One of the patient' s brothers and a nephew showed a similar clinical presentation. Because of the fact that several members of the patient's family suffered from similar symptoms, we consider a syndromic link between XRI and cerebellar disorder to be possible.

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.

The skeletal muscle chloride channel in dominant and recessive human myotonia.

Autosomal recessive generalized myotonia (Becker's disease) (GM) and autosomal dominant myotonia congenita (Thomsen's disease) (MC) are characterized by skeletal muscle stiffness that is a result of muscle membrane hyperexcitability. For both diseases, alterations in muscle chloride or sodium currents or both have been observed. A complementary DNA for a human skeletal muscle chloride channel (CLC-1) was cloned, physically localized on chromosome 7, and linked to the T cell receptor beta (TCRB) locus. Tight linkage of these two loci to GM and MC was found in German families. An unusual restriction site in the CLC-1 locus in two GM families identified a mutation associated with that disease, a phenylalanine-to-cysteine substitution in putative transmembrane domain D8. This suggests that different mutations in CLC-1 may cause dominant or recessive myotonia.

Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice.

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

Evidence against a tumour-specific EcoRI RFLP of the c-mos locus.

EcoRI restriction fragment length polymorphisms (RFLP) at the human c-mos locus were analysed in DNAs of normal individuals and tumour patients. Two alleles with fragment lengths of 2.6 kb (A1) and 5.6 kb (A2) respectively were detected. The allele distribution among the tumour group was similar to that of the control group. No difference was found between the allele frequencies in leucocytes and tumour tissue DNA of the same patients.

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).

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.

Determination of a fragment of the c-erbB-2 translational product p185 in serum of breast cancer patients.

Concentrations of a fragment of the c-erbB-2 translational product (p185 fragment) were measured in serum of 70 breast cancer patients, 19 healthy blood donors, and 18 pregnant women using a heterogenic enzyme immunoassay. The serum concentrations of blood donors and pregnant women were below 30 kU/l. Breast cancer patients showed serum concentrations up to 578 kU/l. All 9/70 patients with serum concentrations higher than 30 kU/l had clinical evidence of metastatic disease and the serum levels of all 35/70 patients without metastasis lay within the normal range. From 9/37 patients with p185 overexpression of the primary tumor in immunohistochemical analysis 3/9 patients with metastatic disease had elevated serum levels higher than 30 kU/l. In all, 6/9 patients without metastasis serum levels were below 30 kU/l. The data of the present study suggest that determination of serum p185 fragment concentrations may be useful as a diagnostic tool in postoperative follow-up of breast cancer patients with c-erbB-2 overexpression of the primary tumor.

Alterations of the c-erbB2 gene in human breast cancer.

DNA amplification, RNA overexpression and p185 protein expression of the c-erbB2 oncogene were investigated in 109 cases of breast cancer with the aim of evaluating any correlation between the different methods. A correlation between Southern blotting and immunohistochemical analysis of paraffin-embedded material was found. Thus, amplification of the c-erbB2 oncogene leads to overexpression of the p185 protein. By contrast, no statistical correlation could be shown between RNA overexpression, measured by Northern blotting, and immunohistochemical p185 membrane stainings. It is of special interest that most of the cases that are positive for Northern blotting and negative for immunochemistry are negative for Southern blotting as well. Contradictory findings between RNA overexpression and lack of immunohistochemical staining of p185 give rise to the assumption that a defective protein is encoded, which cannot be incorporated into the substructures of the tumour cell membrane. When screening for point mutations in the transmembrane domain of the c-erbB2 oncogene, no point mutation could be detected, either by using the endonuclease FokI, which cuts at position 2012 (the point mutation in the neu gene of the rat), or by direct sequencing.

Mouse HNF-3/fork head homolog-1-like gene: structure, chromosomal location, and expression in adult and embryonic kidney.

Screening of a mouse kidney cDNA library with a HNF-3/fork head domain probe revealed cDNA Hfh-1L containing the highly conserved fork head DNA-binding domain. The Hfh1L cDNA shows 92.7% homology at the nucleic acid level with the fork head gene HFH-1 from rat. Southern blot analyses demonstrated that the Hfh-1L gene is highly conserved in a wide variety of species, including goldfish and frog. Sequencing the corresponding genomic clone, we found that the Hfh-1L gene is most likely intronless. By interspecific back-cross analysis, the Hfh-1L gene was localized to mouse chromosome 13. In order to analyze the expression pattern of Hfh-1L, we performed Northern blot analyses and revealed a 2.7-kb transcript in adult kidney and stomach. In situ hybridization experiments of adult mouse kidney showed Hfh-1L expression in the outer medulla of the kidney and the transitional epithelium. In light of the significance of a number of fork head genes in early embryonic development, the pattern of expression during murine embryogenesis was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), and Hfh-1L transcripts were detected in mouse embryos at every stage tested from day 10.5 to 16.5 postconception (p.c.) and in the developing metanephros of 14.5- and 15.5-day p.c. embryos. This expression pattern suggests that the Hfh-1L gene is involved in the development of the kidney.

Isolation and characterization of the human forkhead gene FOXQ1.

We have isolated a human genomic and cDNA clone that encodes a protein of 403 amino acids and belongs to the family of the FOX transcription factors (previously called HNF-3/forkhead transcription factors). The 2.7-kb transcript of the human FOXQ1 gene is expressed predominantly in the stomach, trachea, bladder and salivary gland. Additionally, overexpression of human FOXQ1 was shown in colorectal adenocarcinoma and lung carcinoma cell lines. The FOXQ1 gene is located on chromosome 6p23-25. Databank analysis shows 82% homology with the mouse Foxq1 gene (formerly Hfh-1L) and with a revised sequence of the rat FoxQ1 gene (formerly HFH-1). The DNA-binding motif, named HNF-3/forkhead domain, is well conserved, showing 100% identity in human, mouse, and rat. The human protein sequence contains two putative transcriptional activation domains, which share a high amino acid identity with the corresponding mouse and rat domains.

Amplification of the c-erbB oncogene is associated with malignancy in primary tumours of neuroepithelial tissue.

In various primary brain tumours of neuroepithelial tissue recombinant DNA techniques were used to demonstrate changes of the epidermal growth factor receptor gene, which is homologous to the c-erbB oncogene. Twenty-one of 40 grade III/IV tumours, but only 1 of 16 grade I/II tumours were found to contain amplified and/or rearranged c-erbB sequences. This highly significant difference suggest that c-erbB amplification, rearrangement, or both, are important steps in malignant transformation in a subset of patients with neuroepithelial tumours.

Amplification and expression of the epidermal growth factor receptor gene during the progression of neuroepithelial tumours in vivo.

The epidermal growth factor receptor (EGFR) gene is homologous to the oncogene c-erbB. The occurrence of amplification and rearrangements at the EGFR gene locus is associated with malignancy in neuroepithelial tumours. Sixteen neuroepithelial tumours from eight patients with recurrence of their neoplasms were analysed for changes at the EGFR gene locus and for expression of EGFR. Ten tumours from five patients lacked changes at the EGFR gene locus. Three of eight individuals showed EGFR gene amplifications in both tumours with a higher grade of amplification in the second tumour. In addition to amplification, a rearrangement was found in both tumours of the first patient. In the second case an amplification of chromosome-7-specific c-met sequences was found in the regrown tumour, suggesting that a polysomy 7 was at least partly responsible for the higher copy number of the EGFR sequences. In both tumours of the third patient with EGFR gene amplification different alleles were amplified. In contrast to the findings at the DNA level the EGFR expression, analysed by immunohistochemical techniques, showed a more heterogeneous pattern after tumour progression.

Distribution of epidermal growth factor receptor gene amplification in brain tumours and correlation to prognosis.

In 75 gliomas and 31 meningiomas, mutations at the epidermal growth factor receptor (EGFR) gene locus were restricted to gliomas. The ligands of this receptor, epidermal growth factor and transforming growth factor alpha, lacked quantitative changes at their loci in gliomas and meningiomas. EGFR gene amplification occurred in astrocytomas, oligodendrogliomas, ependymomas and glioblastomas. The frequency of this mutation significantly increased with the malignancy grade and the patient's age. Especially in glioblastomas of individuals aged over 64 years, EGFR gene mutations were observed without chromosome-10-specific allele losses. This finding contradicts the hypothesis that deletion of one entire chromosome 10 regularly precedes EGFR gene amplification in primary glioblastomas of patients aged over 50 years. It was found that most individuals whose gliomas carry an EGFR gene mutation have a poor prognosis, comparable to that of glioblastoma patients even when the tumour is graded as benign.

Prenatal diagnosis of congenital nephrosis of the Finnish type (CNF) in the second trimester.

Congenital nephrosis of the Finnish type is an hereditary, autosomal recessive disease which leads to death in early infancy. This is a case report concerning an affected fetus with legal interruption in the 24th week of gestation on the basis of certain sonographic changes in the fetal kidneys and changes in the protein profile in amniotic fluid, which were consistent with nephrotic damage of the kidneys. Light and electron microscopy showed evidence of CNF, i.e. increase of mesangial matrix and cells in glomeruli, dilated tubular segments, and effaced and plumb foot-processes of the glomerular epithelial cells. Antenatal diagnosis of CNF therefore seems feasible in the second trimester of gestation by means of AFP determinations in maternal serum and amniotic fluid as well as by using sonographic criteria and determination of proteins in amniotic fluid.

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.

Fetal alcohol syndrome in association with Rett syndrome.

Fetal alcohol syndrome in association with RETT syndrome: We report on a girl with neonatal dystrophy, microcephaly, heart defect, and the characteristic features of alcohol embryopathy. Later, she developed distinctive features of RETT syndrome including loss of early acquired developmental skills and presented typical symptoms of RETT syndrome as reduction of communication skills, reduction of hand function, hyperventilation, and grinding of teeth. Molecular analysis of the MECP2 gene revealed the c.808T>C (R270X) mutation located in the nuclear localisation signal sequence of the gene. Our report highlights the importance of considering the diagnosis of RETT syndrome even in patients who are already suffering from a defined disease.