[Holt-Oram syndrome: characterization of a novel mutation]. / Síndrome de Holt-Oram: caracterización de una nueva mutación.

INTRODUCTION: Cardiomyelic syndromes encompass congenital heart disease and skeletal malformations of the upper limbs and are related to mutations in transcription factors with T-Box domains. Holt-Oram syndrome is caused by a dominant mutation in the TBX5 gene that alters the three-dimensional structure of the protein and its DNA binding function. Several point mutations and deletions in TBX5 have been reported in patients with the Holt-Oram syndrome phenotype. PATIENTS AND METHODS: The proband was a boy with a large atrial septal defect ostium secundum type and a ventricular septal defect, diagnosed by clinical findings (heart murmur) and echocardiography. He also presented slightly hypoplastic thumbs with distal bilateral placement and an implantation index of 0.19 (compared with an average of 0.50 for his gestational age at birth). The boy was referred to the department of medical genetics to rule out 22q11.2 microdeletion syndrome. RESULTS: Karyotype and fluorescence in situ hybridization at locus D22S75 were both normal. Because of his clinical findings, molecular study for Holt-Oram syndrome was indicated, leading to the finding of a mutation at intron 7 of TBX5, probably producing a splicing alteration of the gene and resulting in a protein truncated at its C-terminal end. The proband's parents presented the wild type sequence of the gene, thus indicating that the mutation was produced de novo, although a possible germinal mosaicism in the parents could not be ruled out. CONCLUSIONS: Holt-Oram syndrome is the most frequent cause of cardiomyelic syndrome. All children with heart malformations and abnormalities of the upper limbs such as absent, hypoplastic, distally placed or triphalangic thumbs should undergo molecular studies for this syndrome.

Síndrome de Holt-Oram: caracterización de una nueva mutación / Holt-Oram syndrome: characterization of a novel mutation

Introducción Los síndromes cardiomiélicos comprenden cardiopatías congénitas y malformaciones esqueléticas de los miembros superiores, y están relacionados con mutaciones deletéreas de factores de transcripción con dominios del tipo T-Box. El síndrome de Holt-Oram se debe a una mutación dominante en el gen TBX5 que altera la estructura tridimensional de la proteína impidiendo su correcta unión al ADN. Se han descrito varias mutaciones puntuales y deleciones de TBX5 en pacientes con fenotipo de síndrome de Holt-Oram. Pacientes y métodos El paciente es un niño con una comunicación interauricular (CIA) del tipo ostium secundum grande y una comunicación interventricular (CIV) diagnosticados por clínica (soplo) y ecocardiografía. Presenta además unos dedos pulgares algo hipoplásicos y con un emplazamiento distal bilateral, con un índice de implantación de 0,19 frente a una media normal de 0,50 para su edad gestacional al nacer. Es remitido a la consulta de Genética para descartar microdeleción 22q11.2. Resultados El cariotipo y la hibridación in situ de fluorescencia (FISH) con sonda D22S75 resultaron normales y debido a los hallazgos clínicos se realizó un estudio molecular para el síndrome de Holt-Oram. Se encontró una mutación en el intrón 7 de TBX5 que produce una probable alteración del splicing del gen que da lugar a una proteína truncada en su extremo C-terminal. Los padres del propósito presentan una secuencia normal para el gen, lo que indica que la mutación se produjo de novo, sin que pueda descartarse un mosaicismo germinal en los padres. Conclusiones El síndrome de Holt-Oram es la causa más frecuente de síndrome cardiomiélico. Debería ser objeto de estudio molecular todo niño con malformaciones cardíacas y alteraciones de las extremidades superiores como pulgares ausentes, hipoplásicos, distalmente emplazados o trifalángicos

Introduction Cardiomyelic syndromes encompass congenital heart disease and skeletal malformations of the upper limbs and are related to mutations in transcription factors with T-Box domains. Holt-Oram syndrome is caused by a dominant mutation in the TBX5 gene that alters the three-dimensional structure of the protein and its DNA binding function. Several point mutations and deletions in TBX5 have been reported in patients with the Holt-Oram syndrome phenotype. Patients and methods The proband was a boy with a large atrial septal defect ostium secundum type and a ventricular septal defect, diagnosed by clinical findings (heart murmur) and echocardiography. He also presented slightly hypoplastic thumbs with distal bilateral placement and an implantation index of 0.19 (compared with an average of 0.50 for his gestational age at birth). The boy was referred to the department of medical genetics to rule out 22q11.2 microdeletion syndrome. Results Karyotype and fluorescence in situ hybridization at locus D22S75 were both normal. Because of his clinical findings, molecular study for Holt-Oram syndrome was indicated, leading to the finding of a mutation at intron 7 of TBX5, probably producing a splicing alteration of the gene and resulting in a protein truncated at its C-terminal end. The proband's parents presented the wild type sequence of the gene, thus indicating that the mutation was produced de novo, although a possible germinal mosaicism in the parents could not be ruled out. Conclusions Holt-Oram syndrome is the most frequent cause of cardiomyelic syndrome. All children with heart malformations and abnormalities of the upper limbs such as absent, hypoplastic, distally placed or triphalangic thumbs should undergo molecular studies for this syndrome

Euchromatic variant 16p+. Implications in prenatal diagnosis.

BACKGROUND: Euchromatic imbalances at the cytogenetic level are usually associated with phenotypic consequences. Among the exceptions are euchromatic variants of chromosome 16 (16p+) with normal phenotype. There is a growing list of euchromatic duplications and deletions involving both G-positive and G-negative bands that seem to be phenotypically neutral, but these euchromatic variants are rare. OBJECTIVE: The aim of this report is to describe a new familial case of euchromatic variant 16p+ and to emphasise the misinterpretation of these rare euchromatic variants particularly when ascertained at prenatal diagnosis. METHODS AND RESULTS: Fluorescence in situ hybridisation with clone RP11-261A7 showed an amplified signal in the larger chromosome 16. This clone contains FLJ43855 gene, similar to sodium- and chloride-dependent creatine transporter. CONCLUSION: So, this 16p+ variant that involves amplification of pseudogenetic sequences is considered a polymorphism in normal individuals.

MLPA vs multiprobe FISH: comparison of two methods for the screening of subtelomeric rearrangements in 50 patients with idiopathic mental retardation.

Subtelomeric rearrangements not visible by conventional cytogenetic analysis have been reported to occur in approximately 5% of patients with unexplained mental retardation (MR). As the prevalence of MR is high, many patients need to be screened for these chromosomal abnormalities routinely. Multiplex ligation-dependent probe amplification (MLPA) is a new technique for measuring sequence dosage, allowing large number of samples to be processed simultaneously and thus significantly reducing laboratory work. We have assessed its performance for the detection of subtelomeric rearrangements by comparing the results with those of our previous multiprobe fluorescence in situ hybridization (FISH) assay. We have tested 50 patients with idiopathic MR, dysmorphic features, congenital malformations, and/or familial history of MR. Our results show a high degree of concordance between the two techniques for the 50 samples tested. On the basis of these results, we conclude that MLPA is a rapid, accurate, reliable, and cost-effective alternative to FISH for the screening of subtelomeric rearrangements in patients with idiopathic MR.

Comparative study of three diagnostic approaches (FISH, STRs and MLPA) in 30 patients with 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome is commonly diagnosed using fluorescence in situ hybridization (FISH) with commercial probes. The chromosomal breakpoints and deletion size are subsequently characterized by short tandem repeat (STR) segregation tests or by further FISH probes. Recently, a multiplex ligation-dependent probe amplification (MLPA) single tube assay was developed to detect deletions of the 22q11.2 region and other chromosomal regions associated with DiGeorge/velocardiofacial syndrome. We have compared the results of these three techniques in a group of 30 patients affected with 22q11.2 deletion syndrome. MLPA correctly called all patients who had been previously diagnosed by FISH. The MLPA results were concordant in all patients with the STR analysis in respect to deletion size. Furthermore, this novel technique resolved seven cases that were undetermined by STR analysis. These results confirm the efficiency of MLPA as a rapid, reliable, economical, high-throughput method for the diagnosis of 22q11.2 deletion syndrome.

El síndrome de Costello a propósito de un caso clínico / Costello syndrome based on one clinical case

El síndrome de Costello ha sido descrito en 115 pacientes hasta la fecha. Reúne una serie de características fenotípicas que sugieren el diagnóstico ya que, en el momento actual, no existe ningún estudio que lo confirme. El interés de este caso radica en el reconocimiento de este síndrome debido al importante incremento de riesgo tumoral que presentan estos pacientes y a que, por tanto, precisan ser sometidos a un protocolo de despistaje de neoplastias: ecografía abdomino-pélvica desde el nacimiento y cada 3-6 meses hasta 8-10 años con objeto de detectar rabdomiosarcoma; determinación de catecolaminas en orina cada 6-12 meses y hasta los 5 años de vida para objetivar neuroblastoma y búsqueda de hematuria por su relación con la presentaciónde cáncer de vejiga (AU)

Costello Síndrome has been described in 115 patients. It consists in different clinical data that suggest the diagnosis. It is important to recognize this syndrome early because these children have an important tumoral risk so they should undergo different tests in order to find neoplasias: an abdo-pelvic ECO should be done from birth till 8-10 years of age every months to look for rhabdomyosarcoma, urinary catecholamine excretion measurements every 6-12 months up the age of 5 years to find neuroblastoma, and annual screening for hematuria from the age of ten onwards because it is related with the bladder cancer (AU)

Tetrasomy 8p: discordance of amniotic fluid and blood karyotypes.

We describe a girl with congenital heart defect (ventricular septal defect), facial, ear and bone anomalies, agenesis of corpus callosum and conventional cytogenetic studies showing tetrasomy 8p. The identity of the isochromosome was confirmed by fluorescent in situ hybridization (FISH) using painting, subtelomeric and alpha satellite probes for chromosome 8. The extra isochromosome was observed in 100% of cultured peripheral lymphocytes (47,XX,+i(8)(p10)), but normal chromosomes were recorded in cultured amniotic fluid. Microsatellites analysis of the patient's DNA with two markers mapping 8p showed three different peaks, and two markers mapping 8q showed two peaks. To the best of our knowledge, this patient represents the twelfth reported case of tetrasomy 8p. In addition, our report is the first case with a pure tetrasomy 8p in blood, (the other published cases are mosaic 8p), and the second case with a discordance of amniotic fluid and blood karyotypes [Robinow et al., 1989: Am J Med Genet 32:320-324].

Identification of human candidate genes for male infertility by digital differential display.

Evidence for the importance of genetic factors in male fertility is accumulating. In the literature and the Mendelian Cytogenetics Network database, 265 cases of infertile males with balanced reciprocal translocations have been described. The candidacy for infertility of 14 testis-expressed transcripts (TETs) were examined by comparing their chromosomal mapping position to the position of balanced reciprocal translocation breakpoints found in the 265 infertile males. The 14 TETs were selected by using digital differential display (electronic subtraction) to search for apparently testis-specific transcripts in the TIGR database. The testis specificity of the 14 TETs was further examined by reverse transcription-polymerase chain reaction (RT-PCR) on adult and fetal tissues showing that four TETs (TET1 to TET4) were testis-expressed only, six TETs (TET5 to TET10) appeared to be differentially expressed and the remaining four TETs (TET11 to TET14) were ubiquitously expressed. Interestingly, the two tesis expressed-only transcripts, TET1 and TET2, mapped to chromosomal regions where seven and six translocation breakpoints have been reported in infertile males respectively. Furthermore, one ubiquitously, but predominantly testis-expressed, transcript, TET11, mapped to 1p32-33, where 13 translocation breakpoints have been found in infertile males. Interestingly, the mouse mutation, skeletal fusions with sterility, sks, maps to the syntenic region in the mouse genome. Another transcript, TET7, was the human homologue of rat Tpx-1, which functions in the specific interaction of spermatogenic cells with Sertoli cells. TPX-1 maps to 6p21 where three cases of chromosomal breakpoints in infertile males have been reported. Finally, TET8 was a novel transcript which in the fetal stage is testis-specific, but in the adult is expressed in multiple tissues, including testis. We named this novel transcript fetal and adult testis-expressed transcript (FATE).

Cryptic 6q subtelomeric deletion associated with a paracentric inversion in a mildly retarded child.

We report on a girl with minor anomalies and developmental delay carrying an apparently balanced paracentric inversion of chromosome 6q (q22qter). Fluorescent in situ hybridization analysis demonstrated a deletion of the subtelomeric region of 6q. This illustrates the use of specific subtelomeric fluorescent in situ hybridization probes to detect cryptic deletions as an important cause of mental retardation in seemingly balanced chromosome rearrangements.

Molecular characterisation of partial chromosome 21 aneuploidies by fluorescent PCR.

Although trisomy of chromosome 21 is the most prevalent human genetic disorder, data from partial 21 aneuploidies are very scanty. Eight different partial aneuploidies for chromosome 21 were characterised by fluorescence quantitative PCR. Allelic dosage analysis was performed for each patient using 25 CHLC STRs covering the entire q arm. The length of the corresponding trisomies and monosomies was ascertained for five partial trisomics and three partial monosomics. All trisomic patients carried unbalanced translocations involving chromosome 21, whereas one of the monosomic patients bore a ring chromosome 21 and another showed an interstitial deletion of chromosome 21. The chromosomal breakpoints of two partial trisomy patients could be clearly delimited. However, the other three trisomies involved most of the 21 q arm as three allelic doses were detected for each marker. Although these latter patients do not show all the features of Down syndrome, genotype/phenotype correlations agree with previously reported data. The chromosomal breakpoints observed in two partially monosomic patients helped further to define the region involved in different phenotypic features associated with chromosome 21 monosomy. Telomeric material loss was also detected in a patient bearing a ring 21 chromosome. The parental origin of the aneuploidy was assigned for each case, which allowed us to conclude that two of the monosomic cases originated from de novo chromosomal rearrangements. There was no correlation with parental sex in contrast to trisomic patients originating from meiotic nondisjunction.

Characterization of a germline mosaicism in families with Lowe syndrome, and identification of seven novel mutations in the OCRL1 gene.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by major abnormalities of eyes, nervous system, and kidneys. Mutations in the OCRL1 gene have been associated with the disease. OCRL1 encodes a phosphatidylinositol 4, 5-biphosphate (PtdIns[4,5]P2) 5-phosphatase. We have examined the OCRL1 gene in eight unrelated patients with OCRL and have found seven new mutations and one recurrent in-frame deletion. Among the new mutations, two nonsense mutations (R317X and E558X) and three other frameshift mutations caused premature termination of the protein. A missense mutation, R483G, was located in the highly conserved PtdIns(4,5)P2 5-phosphatase domain. Finally, one frameshift mutation, 2799delC, modifies the C-terminal part of OCRL1, with an extension of six amino acids. Altogether, 70% of missense mutations are located in exon 15, and 52% of all mutations cluster in exons 11-15. We also identified two new microsatellite markers for the OCRL1 locus, and we detected a germline mosaicism in one family. This observation has direct implications for genetic counseling of Lowe syndrome families.

Systematic characterisation of disease associated balanced chromosome rearrangements by FISH: cytogenetically and genetically anchored YACs identify microdeletions and candidate regions for mental retardation genes.

Disease associated balanced chromosome rearrangements (DBCRs) have been instrumental in the isolation of many disease genes. To facilitate the molecular cytogenetic characterisation of DBCRs, we have generated a set of >1200 non-chimeric, cytogenetically and genetically anchored CEPH YACs, on average one per 3 cM, spaced over the entire human genome. By fluorescence in situ hybridisation (FISH), we have performed a systematic search for YACs spanning translocation breakpoints. Patients with DBCRs and either syndromic or non-syndromic mental retardation (MR) were ascertained through the Mendelian Cytogenetics Network (MCN), a collaborative effort of, at present, 270 cytogenetic laboratories throughout the world. In this pilot study, we have characterised 10 different MR associated chromosome regions delineating candidate regions for MR. Five of these regions are narrowed to breakpoint spanning YACs, three of which are located on chromosomes 13q21, 13q22, and 13q32, respectively, one on chromosome 4p14, and one on 6q25. In two out of six DBCRs, we found cytogenetically cryptic deletions of 3-5 Mb on one or both translocation chromosomes. Thus, cryptic deletions may be an important cause of disease in seemingly balanced chromosome rearrangements that are associated with a disease phenotype. Our region specific FISH probes, which are available to MCN members, can be a powerful tool in clinical cytogenetics and positional cloning.

Origin of nondisjunction in trisomy 8 and trisomy 8 mosaicism.

Causes of chromosomal nondisjunction is one of the remaining unanswered questions in human genetics. In order to increase our understanding of the mechanisms underlying nondisjunction we have performed a molecular study on trisomy 8 and trisomy 8 mosaicism. We report the results on analyses of 26 probands (and parents) using 19 microsatellite DNA markers mapping along the length of chromosome 8. The 26 cases represented 20 live births, four spontaneous abortions, and two prenatal diagnoses (CVS). The results of the nondisjunction studies show that 20 cases (13 maternal, 7 paternal) were probably due to mitotic (postzygotic) duplication as reduction to homozygosity of all informative markers was observed and as no third allele was ever detected. Only two cases from spontaneous abortions were due to maternal meiotic nondisjunction. In four cases we were not able to detect the extra chromosome due to a low level of mosaicism. These results are in contrast to the common autosomal trisomies (including mosaics), where the majority of cases are due to errors in maternal meiosis.

Fragile X syndrome with extra microchromosome.

A mentally retarded male with Martin-Bell syndrome, who has an extra microchromosome and is fra X negative in cytogenetic study is reported. Because of its small size, the origin of the microchromosome could not be determined. Two other affected males in this family (a cousin and a nephew of the proband) were fragile X positive, 24% and 26%, respectively. Cytogenetic studies and DNA analysis with the probe St B 12.3 were performed on several members of the family. The proband and the two other affected males showed a similar full mutation on the molecular study. This study emphasizes the importance of molecular analysis in the diagnosis of fragile X syndrome, particularly when cytogenetic studies demonstrate fra X negative in individuals in families likely to have X-linked mental retardation.

De novo t(5p;21q) in a patient previously diagnosed as monosomy 21.

In situ hybridization was used to characterize an undetected chromosome translocation in a child whose metaphase chromosome analysis in peripheral blood and in skin culture revealed apparent monosomy 21. The cytogenetic study revealed 45 chromosomes, and no other structural anomalies were detected with G banding. In situ hybridization of chromosome 21-specific probes to metaphase chromosomes and reverse banding from the proband showed a de novo translocation between chromosome 5 and chromosome 21.

A malformed child with a recombinant chromosome 7, rec(7) dup p, derived from a maternal pericentric inversion inv(7)(p15q36).

We report a child with facial dysmorphic features, hypoplasia of the external genitalia, intestinal malrotation, congenital cardiac defect, and minor limb anomalies. Chromosome studies showed a recombinant chromosome 7, rec(7) dup p, resulting from a maternal pericentric inversion inv(7)(p15 q36). Thus, this child had partial trisomy 7p in addition to a small distal monosomy 7. The clinical findings are compared with those found in previous reports of trisomy 7p. Finally, some general principles for genetic counselling are discussed.

Tetraploidy in a liveborn infant.

We report a 3 month old boy with tetraploidy, found in peripheral blood and skin fibroblast cultures, with severely delayed growth and neurodevelopment, and with a cleft lip; these findings have not been described before. This report brings to seven the total number of liveborn infants with a 92,XXYY karyotype.

Balanced reciprocal translocation (X;20) limited to Wilms' tumor in a Wiedemann-Beckwith syndrome.

A girl aged 4 years 3 months with sporadic unilateral Wilms' tumor associated with Wiedemann-Beckwith syndrome, but without aniridia, was found to have a t(X;20) in the tumor cells. Karyotypes of peripheral blood of the patient and her parents were normal. This translocation was confined to the tumor and not been previously reported either in nephroblastoma or any other neoplastic processes. Although there is no microscopic deletion on chromosome 11 and catalase activity was not decreased, we cannot rule out the possibility of a point mutation or a submicroscopic deletion.

The 18p- syndrome. Report of five cases.

Five children, three males and two females were found to have a short arm deletion of chromosome 18. Four of them display some of the typical features of this syndrome: microcephaly, round face, hypertelorism, broad-based nose, "carp-mouth", microrethrognathia, pterygium colli, dysplastic and low set ears, clinodactily, failure to grow, muscular hypotony and mental retardation. Different hypotheses are discussed in order to explain the variable phenotypical expression of the 18 p-syndrome.