Mosaic maternal uniparental isodisomy for chromosome 7q21-qter.

Uniparental disomy (UPD) for several human chromosomes is associated with clinical abnormalities. We report the case of a 2-year-old boy with severe intrauterine and post-natal growth retardation (IUGR/PNGR) and highly variable sweat chloride concentrations. The patient was identified as heterozygous for the F508del mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Unexpectedly, the signal corresponding to the maternally inherited F508del allele appeared much more intense than the paternally derived wild allele. Molecular analysis including polymorphic marker studies, microsatellites and single-nucleotide polymorphisms subsequently showed that the boy was a carrier of a de novo mosaic maternal isodisomy of a chromosome 7 segment while there was a biparental inheritance of the rest of the chromosome. This is the first report of a mosaic partial UPD7. The matUPD7 segment at 7q21-qter extends for 72.7 Mb. The karyotype (550 bands) of our patient was normal, and fluorescence in situ hybridization with probes mapping around the CFTR gene allowed us to rule out a partial duplication. The detection of this chromosomal rearrangement confirms the hypothesis that the 7q31-qter segment is a candidate for the localization of human imprinted genes involved in the control of IUGR and PNGR. It also emphasizes the importance of searching for UPD7 in severe, isolated and unexplained IUGR and PNGR.

Trisomy 22 with thyroid isthmus agenesis and absent gall bladder.

This manuscript reports a fetus of 24 weeks gestation, detected on echography to have congenital anomalies: intra-uterine growth retardation, facial dysmorphism, ventricular septal defect with aortic displacement and 8-mm nuchal skinfold thickness. Karyotype was performed. Post termination of pregnancy autopsy showed additionnal internal organ anomalies included: absent gall bladder and thyroid isthmus agenesis. To our knowledge, these anomalies have never been described in trisomic 22 fetuses. This case suggests that chromosome 22 could play a role in thyroid development.

Pregnancy outcome and prognosis in fetuses with increased first-trimester nuchal translucency.

OBJECTIVE: One of the concerns of prenatal diagnosis is to find sensitive markers to screen for chromosome abnormalities, such as serum assays or nuchal translucency (NT). This study reports our experience with NT measurement during the first trimester of pregnancy. MATERIALS: The study was performed prospectively on 252 fetuses with either NT > or =3 mm or cystic hygroma. RESULTS: We observed 50 abnormal karyotypes, i.e. 19.8%. The incidence of chromosome abnormalities increased with increasing maternal age and increasing NT thickness. For the 202 fetuses with normal karyotypes, outcome was unfavourable in 32 cases: 23 elective terminations of pregnancy, 8 spontaneous abortions and 1 neonatal death. Outcome was favourable in 141 cases. Twenty-nine pregnancies were lost to follow-up. CONCLUSION: Measurement of NT at 12 weeks' gestation seems to be a good marker for chromosome abnormalities. When the karyotype is normal, the pregnancy outcome remains correlated with the degree of NT thickness. The finding of NT >3 mm between 10 and 14 weeks' gestation dictates rigorous ultrasound monitoring and caution when predicting pregnancy outcome.

[Interphase FISH analysis of frozen or fixed tissues for the detection of t(11;14) (q13;q32) in mantle cell lymphoma]. / Analyse par FISH interphasique de tissus congelés ou fixés pour la détection de la t(11;14)(q13;q32) dans les lymphomes du manteau.

Cytogenetic analyses have revealed that mantle cell lymphomas (MCL) are closely associated with the t(11;14)(q13;q32). This translocation juxtaposes the immunoglobulin heavy chain gene (IGH) sequences with the BCL-1 locus, leading to up-regulation of the CCND1 gene and consequently to an overexpression of cyclin D1 protein. We studied 27 MCL with characteristic morphological and immunological (CD5+, CD10-, CD20+, CD23-) features and 2 controls (reactionnal lymphadenitis) to evaluate the feasibility and the interest of FISH analysis on interphase cells from frozen or paraffin-embedded tissues. Sections (CC) and touch preparations (EC) of frozen tissues and sections of paraffin-embedded tissues (CF) were successfully hybridized with the Vysis LSI IgH/CCND1 dual color dual fusion translocation probe. The touch preparations presented a lower cellularity than sections, therefore allowing an easier analysis. Hybridization spots intensities were found stronger in CC and EC than in CF. The percentages of t(11;14) positive cells were similar in CC, EC and CF from a same patient. The percentage of non hybridized cells, analogous in CC and EC, was higher in CF. However, the CF were directly analysed on microscope without the need of any numerical picture treatment. The t(11;14) was detected in all the cases (27/27) and positive cells percentages were always higher than the probe cut-off (5%). The FISH analysis on interphase cells appears a performing and rapid technique to detect t(11;14) in MCL on both frozen and paraffin-embedded tissue, thus extending its practical and diagnostic use.

Gene therapy of a mouse model of protoporphyria with a self-inactivating erythroid-specific lentiviral vector without preselection.

Successful treatment of blood disorders by gene therapy has several complications, one of which is the frequent lack of selective advantage of genetically corrected cells. Erythropoietic protoporphyria (EPP), caused by a ferrochelatase deficiency, is a good model of hematological genetic disorders with a lack of spontaneous in vivo selection. This disease is characterized by accumulation of protoporphyrin in red blood cells, bone marrow, and other organs, resulting in severe skin photosensitivity. Here we develop a self-inactivating lentiviral vector containing human ferrochelatase cDNA driven by the human ankyrin-1/beta-globin HS-40 chimeric erythroid promoter/enhancer. We collected bone marrow cells from EPP male donor mice for lentiviral transduction and injected them into lethally irradiated female EPP recipient mice. We observed a high transduction efficiency of hematopoietic stem cells resulting in effective gene therapy of primary and secondary recipient EPP mice without any selectable system. Skin photosensitivity was corrected for all secondary engrafted mice and was associated with specific ferrochelatase expression in the erythroid lineage. An erythroid-specific expression was sufficient to reverse most of the clinical and biological manifestations of the disease. This improvement in the efficiency of gene transfer with lentiviruses may contribute to the development of successful clinical protocols for erythropoietic diseases.

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells.

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fech(m1Pas)/Fech(m1Pas) murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Leucodystrophy and oculocutaneous albinism in a child with an 11q14 deletion.

We report a patient with an undetermined leucodystrophy associated with type 1A oculocutaneous albinism (OCA). Type 1 OCA results from recessive mutations in the tyrosinase gene (TYR) located in 11q14.3. The patient was found by FISH to carry a deletion of at least the first exon of the TYR gene on one chromosome and a (TG) deletion at codon 244/245 on the second chromosome. The existence of the microdeletion suggested that a gene responsible for leucodystrophy was located in the vicinity of the TYR gene. A combination of a test of hemizygosity and contig mapping studies allowed us to map the gene within a 0.6 cM region flanked by microsatellite markers D11S1780 and D11S931.

Case with autistic syndrome and chromosome 22q13.3 deletion detected by FISH.

Autism is a rare neurodevelopmental disorder with a strong genetic component. Co-occurrence of autism and chromosomal abnormalities is useful to localize candidate regions that may include gene(s) implicated in autism determinism. Several candidate chromosomal regions are known, but association of chromosome 22 abnormalities with autism is unusual. We report a child with autistic syndrome and a de novo 22q13.3 cryptic deletion detected by FISH. Previously described cases with 22q13.3 deletions shared characteristic developmental and speech delay, but autism was not specifically reported. This case emphasizes a new candidate region that may bear a gene involved in autism etiopathogenesis. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:839-844, 2000.

Mapping, characterization, and expression analysis of the SM-20 human homologue, c1orf12, and identification of a novel related gene, SCAND2.

Major psychosis was shown to segregate with a balanced translocation (1q42.1; 11q14.3) in a multigenerational family. This study describes the identification of a human SM-20 homologue gene that lies at about 400 kb on the centromeric side of the 1q42.1 breakpoint. The full-length cDNA sequence and gene structure were determined. Expression analysis was performed, showing high expression levels in skeletal and cardiac muscles; in the central nervous system, expression was restricted to dopaminergic neurons and spinal motoneurons. A second gene displaying high sequence similarity with SM-20 was also identified by BLAST. This gene, located on chromosome 15, is likely to have evolved by retroposition of SM-20 mRNA and an exon-shuffling mechanism. It encodes a 306-amino-acid protein harboring strong homology with an N-terminal motif found in some zinc-finger proteins. This gene was named SCAND2 (SCAN domain-containing 2).

[Williams syndrome (microdeletion 7q11.23), model of behavioral phenotype]. / Le syndrome de Williams (microdélétion 7q11.23), modèle de phénotype comportemental.

A specific behavioral phenotype is recognized in some genetic entities, particularly in microdeletion syndromes secondary to cytogenetically undetectable chromosomal deletions. Williams syndrome (WS) is a developmental disorder displaying dysmorphic signs, heart malformations and behavioral phenotype associated, in most cases, with a deletion of chromosome 7q11.23. We described physical and neuro-psychological assessment of nine cases of SW. Molecular studies were performed using Southern blot analyses or FISH, and identified a 7q11.23 deletion in all cases. Behavioral phenotype of WS is characterized by hyperactivity, engaging and jovial personality, hypersensitivity in hearing, and some elements of speech may be enhanced. Moderate mental retardation is frequently present. Previously reported studies have revealed specific cognitive deficits including deficits in language development and in long term memory, and poor visual-motor integration. Two genes, LIMK1 and STX1A, have been supposed to be implicated in determinism of WS behavioral phenotype. A specific behavioral phenotype is also demonstrated in others microdeletion syndromes, focusing attention on some chromosomal regions supposed to contain candidate genes involved in cognitive and behavioral traits.

Reversion of hepatobiliary alterations By bone marrow transplantation in a murine model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is characterized clinically by cutaneous photosensitivity and biochemically by the accumulation of excessive amounts of protoporphyrin in erythrocytes, plasma, feces, and other tissues, such as the liver. The condition is inherited as an autosomal dominant or recessive trait, with a deficiency of ferrochelatase activity. A major concern in EPP patients is the development of cholestasis with accumulation of protoporphyrin in hepatobiliary structures and progressive cellular damage, which can rapidly lead to fatal hepatic failure. The availability of a mouse model for the disease, the Fech(m1Pas)/Fech(m1Pas) mutant mouse, allowed us to test a cellular therapy protocol to correct the porphyric phenotype. When Fech/Fech mice received bone marrow cells from normal animals, the accumulation of protoporphyrin in red blood cells and plasma was reduced 10-fold but still remained 2.5 times above normal levels. Interestingly, in very young animals, bone marrow transplantation can prevent hepatobiliary complications as well as hepatocyte alterations and partially reverse protoporphyrin accumulation in the liver. Bone marrow transplantation may be an option for EPP patients who are at risk of developing hepatic complications.

High-resolution integrated map encompassing the breast cancer loss of heterozygosity region on human chromosome 16q22.1.

Loss of heterozygosity (LOH) on the long arm of human chromosome 16 is a common genetic alteration observed in both invasive ductal and invasive lobular breast carcinomas. We have generated a high-resolution integrated map encompassing the smallest region of LOH overlap within chromosome 16q22.1 (SRO2). Southern hybridization experiments using more than 140 probes resulted in the assembly of 152 bacterial large-insert clones into a 2.8-Mb contig covering SRO2. The structure of the contig was verified by long-range mapping using total human genomic DNA, and the contig orientation was determined by fluorescence in situ hybridization. A total of 68 transcripts have been identified in the map. One of the genes residing within SRO2 is the E-cadherin gene, CDH1, which has previously been shown to be mutated in lobular breast carcinomas, resulting in loss of E-cadherin expression. In most cases of ductal carcinoma, which is the major mammary cancer type, E-cadherin is normally expressed, suggesting that other genes within 16q22.1 are involved in the development of this tumor subtype. The high-resolution map presented in this study provides a valuable resource for identification of tumor suppressor genes expected to be involved in the etiology of breast carcinomas.

A novel immunodeficient mouse model--RAG2 x common cytokine receptor gamma chain double mutants--requiring exogenous cytokine administration for human hematopoietic stem cell engraftment.

Gene transduction into immature human hematopoietic cells collected from umbilical cord blood, bone marrow, or mobilized peripheral blood cells could be useful for the treatment of genetic and acquired disorders of the hematopoietic system. Immunodeficient mouse models have been used frequently as recipients to assay the growth and differentiation of human hematopoietic stem/progenitor cells. Indeed, high levels of human cell engraftment were first reported in human/murine chimeras using NOD/SCID mice, which now are considered as the standard for these types of experiments. However, NOD/SCID mice have some clear disadvantages (including spontaneous tumor formation) that limit their general use. We have developed a new immunodeficient mouse model by combining recombinase activating gene-2 (RAG2) and common cytokine receptor gamma chain (gamma c) mutations. The RAG2-/-/gamma c- double mutant mice are completely alymphoid (T-, B-, NK-), show no spontaneous tumor formation, and exhibit normal hematopoietic parameters. Interestingly, human cord blood cell engraftment in RAG2-/-/gamma c- mice was greatly enhanced by the exogenous administration of human cytokines interleukin-(IL-3) granulocyte-macrophage colony-stimulating factor, (GM-CSF), and erythropoietin in contrast to the NOD/SCID model. This unique feature of the RAG2-/-/gamma c- mouse model should be particularly well suited for assessing the role of different cytokines in human lymphopoiesis and stem/progenitor cell function in vivo.

Abdominal lymphatic dysplasia and 22q11 microdeletion.

We report the case of a child with 22q11 microdeletion who presented with abdominal lymphatic dysplasia resulting in exsudative enteropathy. This primitive and localized lymphatic malformation is consistent with the vascular theory in the velocardiofacial syndrome.

Fluorescence-based selection of retrovirally transduced cells in congenital erythropoietic porphyria: direct selection based on the expression of the therapeutic gene.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is an inherited disease caused by a deficiency of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor with death occurring in early adult life and available treatments are only symptomatic and unsatisfactory. In vitro gene transfer experiments have documented the feasibility of gene therapy via haematopoietic stem cells to treat this disease. To facilitate future ex vivo gene therapy in humans, the design of efficient selection procedures to increase the frequency of genetically corrected cells prior to autologous transplantation is a critical step. METHODS: An alternative selection procedure based upon expression of a transferred gene was performed on a lymphoblastoid (LB) cell line from a patient with congenital erythropoietic porphyria to obtain high frequencies of genetically modified cells. The presence of exogeneous delta-aminolevulinic acid (ALA), a haem precursor, induces an increase in porphyrin accumulation in LB deficient cells. Porphyrins exhibit a specific fluorescent emission and can be detected by cytofluorimetry under ultraviolet excitation. RESULTS: In genetically modified cells, the restored metabolic flow from ALA to haem led to a lesser accumulation of porphyrins in the cells, which were easily separated from the deficient cells by flow cytometry cell sorting. CONCLUSION: This selection process represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.