Rashida Anwar

Molecular basis of inherited factor XIII deficiency: identification of multiple mutations provides insights into protein function

Summary. Factor XIII (FXIII) is a zymogen essential for normal haemostasis. In inherited FXIII deficiency the majority of cases show absence of the FXHIa subunit. Molecular analysis of PCR‐amplified FXIIIa subunit exonic regions, and of RT‐PCR amplified cDNA from six patients with FXIIIa subunit deficiency, from five unrelated families, has revealed 10 sequence changes: three mutations resulting in abnormal splicing of pre‐mRNA, one nonsense mutation, one deletion/insertion change, three point mutations producing Val34Leu, Asn60Lys and Arg408Gln changes, and two silent mutations. In three families the patients are homozygous for a specific deficiency causing mutation, and patients from the remaining two families are compound heterozygotes. Understanding the molecular pathology of the disorder provides insights into the structure‐function relationships of the various domains within the FXm protein. From a clinical point of view, it enables direct diagnosis at the DNA level and may aid the development of FXIII analogues to promote wound healing.

Molecular analysis of the presenilin 1 (S182) gene in "sporadic" cases of Alzheimer's disease: identification and characterisation of unusual splice variants.

Abstract: Mutations of the presenilin 1 (PS‐1) gene at the Alzheimers disease (AD) FAD3 locus on chromosome 14q24.3 are responsible for the majority of familial early‐onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in “sporadic” disease, we performed a molecular analysis of PS‐1 transcripts extracted from brain tissues of a series of histologically confirmed cases of “sporadic” AD (n = 10) and also from histologically “normal” (non‐Alzheimer) age‐matched brain controls (n = 5). No sequence changes in the PS‐1 coding sequence were detected after analysis by reverse transcription‐PCR. This suggests that the frequency of mutations in the PS‐1 (S182) coding region in “sporadic” Alzheimers disease is very low. However, we demonstrated that the PS‐1 gene is highly variably spliced. One splice variant involves the 5′ untranslated region of the PS‐1 gene only and hence encodes for normal PS‐1. Six further splice variants involve coding regions of the PS‐1 gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS‐1 gene. One of these variants, resulting in the loss of transmembrane domain TM‐VII, was found only in an AD patient.

Molecular analysis of sixteen unrelated factor XIIIA deficient families from south‐east of Iran

high-risk myeloma. A major goal of our study was to demonstrate reproducible methods to establish various, clinically relevant myeloma cell lines (Li et al, 2007). We have recently described that both hyperdiploid and nonhyperdiploid cases were equally common (Zhan et al, 2006). We also believe that the two cases from which the LD and CF lines were established, derived from advanced hyperdiploid myelomas that turned to a proliferation signature over time. CF cells but not LD cells had partial trisomies associated with hyperdiploid myeloma. Although karyotype analysis did not show the classic trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21, the gene expression profile clearly indicated that many genes overexpressed in these two cases were from those chromosomes (data not shown). This may be due to the sensitive molecular gene expression profiling used to detect the genetic abnormalities. From 351 TT2 dataset, 6% of the myelomas in both hyperdipoid (HY) and proliferation (PR) groups were found in the group showing the lowest 10% TP53 expression (F. Zhan and J. Shaughnessy, unpublished observations), indicating that hyperdiploid myeloma could also harbor 17p13 deletion with low TP53 expression. Furthermore, DKK1, which is typically associated with hyperdiploid myeloma, was highly expressed in both LD and CF cell lines. Although recent studies have identified subtypes of HY myelomas with poor prognosis (Zhan et al, 2006; Chng et al, 2007), high expression of certain genes by LD and CF cells (e.g. CXCR4) and their growth characteristics ex vivo and in our animal models indicate that these cells are highly dependent on the bone marrow microenvironment, a typical feature of hyperdiploid myeloma. We believe that the procedures used in our work will result in the establishment of additional useful hyperdiploid cell lines.

The genetics of primary open angle glaucoma

The advent of the discipline of molecular genetics over the past decade has led to a dramatic growth in our understanding of the genetics of a myriad of diseases. Ophthalmology has benefited greatly from this new technology, with significant advances in our knowledge about conditions as varied as aniridia and retinitis pigmentosa.1 2 Our understanding of the genetics of primary open angle glaucoma (POAG) may not be as clear as with some other ophthalmic conditions but, nevertheless, there have been great advances since the last review about the genetics of glaucoma published in the BJO in 1980.3 At that time, our knowledge was based on a number of conflicting studies attempting to link human polymorphisms, such as the ability to taste phenyl thiocarbamide, with glaucoma.4 Nowadays, the positions of genes responsible for various forms of glaucoma have been localised, not just to individual chromosomes, but to specific small regions on those chromosomes. Recently, for the first time, a gene responsible for a specific form of POAG has been identified. This review aims to highlight and explain the important recent advances in our understanding of the genetics of POAG. Primary open angle glaucoma, for the purpose of this review, refers to those cases of glaucoma in which there is not only no evident antecedent or related ocular disease but also the angle of the anterior chamber remains open at all times.5 The possibility of a genetic predisposition to glaucoma was first realised in 1842 when Benedict reported the occurrence of glaucoma in two sisters.6 Despite the intervening 150 years, our understanding of the genetics of POAG remains unclear. Certainly, most POAG pedigrees do not show a simple Mendelian pattern of inheritance. However, relatives of patients with glaucoma do run an increased risk of developing the condition …

An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas

BackgroundInactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies.MethodsWe analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression.Results50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene.ConclusionsThe results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.

Genetic screening in a large family with juvenile onset primary open angle glaucoma

AIMS A number of genetic loci have been implicated in the pathogenesis of primary open angle glaucoma (POAG). The aim of this study was to identify the genetic cause of POAG in a large Scottish family and, if possible, offer genetic screening and advice to family members. METHODS Family members were examined to determine their disease status. Base excision sequence scanning was carried out in order to test for the presence of a POAG causing mutation at known genetic loci. Direct DNA sequencing was performed in order to determine the mutation sequence. RESULTS All family members of known affected disease status and two family members of unknown disease status were found to have a mutation in theTIGR gene. The mutation resulted in the substitution of a glycine residue with an arginine residue at codon 252 (Gly252Arg). No other sequence variations were present in any members of the family. CONCLUSION The Gly252Arg mutation in the TIGR gene results in the development of POAG in this family. It was possible to identify younger, currently unaffected, members of the family who carry the mutation and who are therefore at a very high risk of developing POAG themselves. This is the first demonstration that Gly252Arg can be a disease causing mutation rather than a benign polymorphism. The possible pathogenic mechanisms and wider implications of the mutation are considered.

Splicing and missense mutations in the human FXIIIA gene causing FXIII deficiency : effects of these mutations on FXIIIA RNA processing and protein structure

We have investigated the molecular basis of factor XIII (FXIII) deficiency in a family from the north‐west region of the U.K. and identified two sequence changes in the FXIII subunit A (FXIIIA) gene. We report a novel Asn to Lys mutation at codon 541, and a g → a mutation at the intron 5/exon 6 splice junction in the FXIIIA gene. The splicing mutation results in two abnormal FXIIIA transcripts. The Asn541 residue is important for stabilizing an external fold in the FXIIIA barrel 1 domain. The Asn541Lys mutation is expected to result in inappropriate folding and therefore an unstable FXIIIA molecule.

Prevalence and clinical challenges among adults with primary immunodeficiency and recombination-activating gene deficiency

To the Editor: Recombination-activating gene (RAG) deficiency has an estimated disease incidence of 1:181,000, including severe combined immunodeficiency (SCID) at a rate of 1:330,000. Complete or hypomorphic variants of SCID secondary to low recombinase activity (<5%) present early with severe infections and/or clinical signs of systemic inflammation, such as severe dermatitis, colitis, or both. Hypomorphic RAG1/2 mutations with more preserved residual V(D)J recombination activity (5% to 30%) result in a distinct phenotype of combined immunodeficiency with granuloma, autoimmunity, or both. Beyond combined immunodeficiency, RAG deficiency has been found in patients with predominantly primary antibody deficiencies and naive CD4 T-cell lymphopenia in most cases. Currently, there is no published systematic evaluation for the presence of an underlying RAG deficiency in patients with primary antibody deficiencies. There is great variability among diagnostic modalities for evaluation and treatment for inflammatory lung disease in case reports of RAG deficiency with no standardized guidelines. Clinical features and lung disease for patients with late presentation of RAG deficiency have not been studied extensively. In addition, no studies have examined the prevalence of RAG deficiency in cohorts of adults with primary immunodeficiency (PID). Here we describe a cohort of 15 patients with late presentation of RAG deficiency. We also estimate the prevalence of RAG deficiency in adults with PID after genetic analysis in 2 separate large cohorts of patients with PID. We have analyzed the canonical regions of RAG1 and RAG2 in a total of 692 patients with PID from 2 separate cohorts, one from the United Kingdom (UK) and one from Austria (Vienna). The UK cohort is part of the National Institute for Health Research BioResource–Rare Diseases PID study, as previously described (Tuijnenburg et al). In the National Institute for Health Research BioResource–Rare Diseases PID cohort of 558 patients (299 adults) and the Vienna cohort of 134 patients (106 adults), we report a total of 5 newly identified cases of RAG deficiency. For details, see the Methods section and Tables E1 to E3 in this article’s Online Repository at www.jacionline.org. Based on these findings, we estimate that the prevalence of RAG deficiency in adults with PID ranges from 1% to 1.9%. For all adult patients with PID currently registered with the UK Primary Immunodeficiency Network database (3294 patients older than age 18 years), we expect to find an additional 32.9 to 62.6 cases of RAG deficiency. Gene variants are shown in Fig 1, A. Cohort demographics are discussed in the Methods section in this article’s Online Repository. Functional characterization of novel RAG variants is discussed in the Methods section in this article’s Online Repository. The activity of mutant RAG1 and RAG2 proteins normally required for catalyzingV(D)J recombination events are shown in Table E2. In addition to the method previously described, we also used a

The Arg703Trp missense mutation in F13A1 is a de novo event

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Bialellic Mutations in Tetratricopeptide Repeat Domain 7A ( TTC7A ) Cause Common Variable Immunodeficiency-Like Phenotype with Enteropathy

TTC7A deficiency typically causes severe gastrointestinal manifestations such as multiple intestinal atresia or early-onset inflammatory bowel disease. In some cases, this is associated with severe combined immunodeficiency. Partial loss-of-function mutations appear to be associated with a milder phenotype resulting in common variable immunodeficiency-like condition with enteropathy.