Defective class II transactivator expression in a B lymphoma cell line.

Loss of MHC class II expression in B-cell lymphoma has been associated with a higher tumorigenicity resulting from lower titers of tumor-infiltrating lymphocytes. This report aims towards the identification of the molecular mechanism leading to defective MHC class II expression in a B-cell lymphoma cell line, Rec-1. We evidenced a coordinated alteration of HLA-D gene transcription, reminiscent of B lymphoblastoid cell lines from patients with MHC class II deficiency. Genetic complementation performed between these cell lines and the lymphoma cells indicated that Rec-1 is altered in the MHC2TA gene. MHC2TA encodes the class II transactivator (CIITA), the master regulator of HLA-D gene expression. However, the coding sequence of the Rec-1 CIITA transcript did not reveal any mutation that could hamper the activity of the encoded protein. In agreement with the genetic complementation analysis, we evidenced a highly residual CIITA protein expression in the Rec-1 cell line resulting from a transcriptional defect affecting MHC2TA expression. Anti-HLA-DR monoclonal antibody treatment has proved efficient in the destruction of B lymphoma cells. Our data indicate that the appearance of variants losing CIITA, and thereby HLA-DR, expression will require a thorough monitoring during such immunotherapy protocols.

[Analysis of hearing impairment causes in molecular diagnosis of deafness]. / Analiza przyczyn uszkodzen sluchu w swietle diagnostyki molekularnej gluchoty.

Deafness is one of the most frequent congenital hearing impairments. Knowledge of its causes will result in elimination of risk factors and applying prophylactic activities. It is recognized that about 40% of hearing impairments have genetic origin and 80% of these are autosomal recessive. Introducing molecular diagnosis to medical practice makes precise identification of hearing impairment and genetic counseling possible. The authors present results of DNA examination in patients with hereditary deafness, performed at the National Research Institute of Mother and Child in Warsaw. Genetic cause of deafness was confirmed in 60% cases.

High frequency of GJB2 gene mutations in Polish patients with prelingual nonsyndromic deafness.

We report an analysis of 102 unrelated Polish patients with profound prelingual deafness for mutations in the GJB2 gene (OMIM #220290). Mutations were found in 41/102 (40%) subjects. Among mutated alleles, 35delG was prevalent and present in 88%. In nine alleles, different mutations were found: M34T, Q47X, R184P, and 313del14 (found in 6 patients). The results prove mutations in the GJB2 gene are responsible for much hereditary nonsyndromic deafness in Poland, with a strong prevalence of the 35delG mutation. We have also found a high carrier frequency (1/50) for the 35delG mutation in the Polish population.

Mutation in the class II trans-activator leading to a mild immunodeficiency.

The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcriptional level. Four trans-acting proteins control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated protein; RFX protein containing ankyrin repeats, and RFX5. In humans, defects in these genes result in MHC class II expression deficiency and cause combined immunodeficiency. Most patients with this deficiency suffer from severe recurrent infections that frequently lead to death during early childhood. We investigated three sisters, now ages 21, 22, and 24 years, in whom MHC-II deficiency was detected. Even though the eldest sibling was asymptomatic and the other two had only mild immunodeficiency, none of the three class II isotypes was expressed on T cell blasts, fibroblasts, EBV B cell lines, or epidermal dendritic cells. Residual HLA-II expression was detected in fresh PBMC. Somatic complementation identified the disease as CIITA deficiency. A homozygous T1524C (L469P) substitution was found in the coding region of the CIITA cDNA and was shown to be responsible for the defect in MHC-II expression. This missense mutation prevents the normal functioning of MHC-II but does not lead to the nuclear exclusion of the L469P CIITA. Transfection experiments demonstrated that the CIITA L469P mutant had residual MHC class II trans activation activity, which might explain the unusual clinical course of the patients studied. This study shows that an attenuated clinical phenotype or an asymptomatic clinical course can be observed in patients despite a profound defect in the expression of MHC class II genes. The frequency of the inherited MHC class II deficiency might thus be underestimated.

In vitro expression analysis of R68G and R68S mutations in phenylalanine hydroxylase gene.

Phenylketonuria (PKU), an autosomal recessive disorder caused be a deficiency of hepatic phenylalanine hydroxylase (PAH), is clinically very heterogeneous. At the molecular level, more than 400 mutations in the PAH gene are known to date, which in different genotype combinations could account for biochemical and clinical variability of symptoms. In vitro expression studies on R68G and R68S mutations causing mild phenylketonuria are presented.

Founder effect for a 26-bp deletion in the RFXANK gene in North African major histocompatibility complex class II-deficient patients belonging to complementation group B.

Expression of major histocompatibility complex (MHC) class II genes is controlled at the transcriptional level by at least four trans-acting genes, CIITA, RFXANK, RFX5, and RFXAP. Defects in these regulatory genes result in the absence of MHC class II molecule expression and, thereby, cause a combined immunodeficiency. MHC class II deficiency is inherited as an autosomal recessive trait. Since the first description of the disease, about 70 patients from 50 families have been reported. Forty-three of these families have been classified into four complementation groups: A, B, C, and D. In the largest group, B, the majority of patients are of North African origin. In two of these patients, the same mutation in the RFXANK gene (752delG-25) was identified. We performed a mutation analysis in 20 additional patients belonging to complementation group B and detected the 752delG-25 mutation in 17. All of these patients are of North African origin. A founder effect for this mutation was documented, since all tested patients, except one, display a common haplotype spanning the RFXANK locus.

A novel hereditary developmental vitreoretinopathy with multiple ocular abnormalities localizing to a 5-cM region of chromosome 5q13-q14.

BACKGROUND: To undertake a clinical and molecular analysis of a previously unpublished kindred with a phenotypically distinct vitreoretinopathy characterized by associated ocular developmental abnormalities. DESIGN: Family genetic study. PARTICIPANTS: A total of 23 members, both affected and unaffected, of 1 kindred with vitreoretinopathy. METHOD: Individuals within the kindred were examined clinically and blood samples taken for DNA analysis. Genetic analysis was performed for the proximal region of chromosome 5q by means of polymerase chain reaction (PCR). MAIN OUTCOME MEASURES: Detection of vitreoretinopathy and associated abnormalities. RESULTS: This novel, hereditary vitreoretinopathy, showing the classic features of vitreous pathology and early-onset retinal detachments, was associated with a variety of ocular developmental abnormalities, including posterior embryotoxon, congenital glaucoma, iris hypoplasia, congenital cataract, ectopia lentis, microphthalmia, and persistent hyperplastic primary vitreous. There were no associated systemic features. Genetic mapping with markers from the proximal region of 5q13-q14 showed linkage to a 5-cM region between the markers D5S626 and D5S2103. CONCLUSIONS: The 5-cM region is within that implicated in the etiology of both Wagner and erosive vitreoretinopathies. This suggests that this novel condition may be allelic, refines the genetic mapping for vitreoretinopathies that map to 5q13-q14, and implicates a gene important not only in vitreous production but also in early ocular development.

Refined genetic and physical localization of the Wagner disease (WGN1) locus and the genes CRTL1 and CSPG2 to a 2- to 2.5-cM region of chromosome 5q14.3.

Wagner syndrome (WGN1; MIM 143200), an autosomal dominant vitreoretinopathy characterized by chorioretinal atrophy, cataract, and retinal detachment, is linked to 5q14.3. Other vitreoretinopathies without systemic stigmata, including erosive vitreoretinopathy, are also linked to this region and are likely to be allelic. Within the critical region lie genes encoding two extracellular macromolecules, link protein (CRTL1) and versican (CSPG2), which are important in binding hyaluronan, a significant component of the mammalian vitreous gel, and which therefore represent excellent candidates for Wagner syndrome. Genetic mapping presented here in two further families reduces the critical region to approximately 2 cM. Subsequent refinement of the physical map allows ordering of known polymorphic microsatellites and excludes CRTL1 as a likely candidate for the disorder. CSPG2 is shown to lie within the critical region; however, analysis of the complete coding region of the mature peptide reveals no clear evidence that it is the gene underlying WGN1.

[Neurofibromatosis type 2 (NF2)--classical example of a rare familial cancer syndrome]. / Nerwiakowlókniakowatosc typu 2 (NF2)--klasyczny przyklad rodzinnego zespolu nowotworowego.

Type 2 neurofibromatosis (NF2) is a dominantly inherited disorder characterized by a predisposition to multiple tumours of the nervous system. The incidence in the Caucasian population is estimated between one in 35,000 and one in 40,000 of live births. The NF2 gene was isolated and shown to have mutations in NF2 patients. It is considered to belong to tumour suppressor gene family. Direct NF2 gene analysis enables verification of tentative clinical diagnosis and makes genetic counselling possible.

[Molecular diagnosis of inherited disease]. / Diagnostyka molekularna chorób uwarunkowanych genetycznie.

The ability to detect mutations in nucleic acids sequence is of fundamental importance in medical molecular genetics. The aim is to link mutations of identified genes to particular disease phenotypes and to facilitate rapid, molecular diagnosis. There are numerous techniques described for screening unknown mutations, and for diagnosing those which have been identified previously. Each method offers different advantages and has its own limitations.

Genetic and molecular definition of complementation group D in MHC class II deficiency.

Four complementation groups, A, B, C and D, have been described among cell lines defective in the coordinate expression of MHC class II genes. These include cell lines established from patients affected with MHC class II deficiency and experimentally generated mutant cell lines. Group D, in contrast to the other groups, was for a long time represented only by the 6.1.6 mutant cell line. The gene responsible for the defect in this group, RFXAP , recently was cloned and found to be mutated in the 6.1.6 cell line and in three patients. Here we report fusion experiments in several new HLA class II-deficient patients, completing the classification of the majority of known patients into the four complementation groups. Patients from five unrelated families were classified in complementation group D, while nine others fall into complementation groups A and B. None of the patients defined a new complementation group. Full correction of MHC class II expression was obtained in cells from patients belonging to group D by transfection with the RFXAP cDNA. The RFXAP coding region was found to be mutated in all patients. Mutations were found to be recurrent since only three different mutations have been found in the eight unrelated families reported to date.