Michał Witt

RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa.

Introduction: Primary ciliary dyskinesia (PCD) is a rare disease classically transmitted as an autosomal recessive trait and characterised by recurrent airway infections due to abnormal ciliary structure and function. To date, only two autosomal genes, DNAI1 and DNAH5 encoding axonemal dynein chains, have been shown to cause PCD with defective outer dynein arms. Here, we investigated one non-consanguineous family in which a woman with retinitis pigmentosa (RP) gave birth to two boys with a complex phenotype combining PCD, discovered in early childhood and characterised by partial dynein arm defects, and RP that occurred secondarily. The family history prompted us to search for an X linked gene that could account for both conditions. Results: We found perfect segregation of the disease phenotype with RP3 associated markers (Xp21.1). Analysis of the retinitis pigmentosa GTPase regulator gene (RPGR) located at this locus revealed a mutation (631_IVS6+9del) in the two boys and their mother. As shown by study of RPGR transcripts expressed in nasal epithelial cells, this intragenic deletion, which leads to activation of a cryptic donor splice site, predicts a severely truncated protein. Conclusion: These data provide the first clear demonstration of X linked transmission of PCD. This unusual mode of inheritance of PCD in patients with particular phenotypic features (that is, partial dynein arm defects and association with RP), which should modify the current management of families affected by PCD or RP, unveils the importance of RPGR in the proper development of both respiratory ciliary structures and connecting cilia of photoreceptors.

Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders - updated European recommendations

The increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.

DNAI2 Mutations Cause Primary Ciliary Dyskinesia with Defects in the Outer Dynein Arm

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic destructive airway disease and randomization of left/right body asymmetry. Males often have reduced fertility due to impaired sperm tail function. The complex PCD phenotype results from dysfunction of cilia of the airways and the embryonic node and the structurally related motile sperm flagella. This is associated with underlying ultrastructural defects that frequently involve the outer dynein arm (ODA) complexes that generate cilia and flagella movement. Applying a positional and functional candidate-gene approach, we identified homozygous loss-of-function DNAI2 mutations (IVS11+1G > A) in four individuals from a family with PCD and ODA defects. Further mutational screening of 105 unrelated PCD families detected two distinct homozygous mutations, including a nonsense (c.787C > T) and a splicing mutation (IVS3-3T > G) resulting in out-of-frame transcripts. Analysis of protein expression of the ODA intermediate chain DNAI2 showed sublocalization throughout respiratory cilia. Electron microscopy showed that mutant respiratory cells from these patients lacked DNAI2 protein expression and exhibited ODA defects. High-resolution immunofluorescence imaging demonstrated absence of the ODA heavy chains DNAH5 and DNAH9 from all DNAI2 mutant ciliary axonemes. In addition, we demonstrated complete or distal absence of DNAI2 from ciliary axonemes in respiratory cells of patients with mutations in genes encoding the ODA chains DNAH5 and DNAI1, respectively. Thus, DNAI2 and DNAH5 mutations affect assembly of proximal and distal ODA complexes, whereas DNAI1 mutations mainly disrupt assembly of proximal ODA complexes.

Recent advances in primary ciliary dyskinesia genetics

Primary ciliary dyskinesia (PCD) is a rare genetically heterogeneous disorder caused by the abnormal structure and/or function of motile cilia. The PCD diagnosis is challenging and requires a well-described clinical phenotype combined with the identification of abnormalities in ciliary ultrastructure and/or beating pattern as well as the recognition of genetic cause of the disease. Regarding the pace of identification of PCD-related genes, a rapid acceleration during the last 2–3 years is notable. This is the result of new technologies, such as whole-exome sequencing, that have been recently applied in genetic research. To date, PCD-causative mutations in 29 genes are known and the number of causative genes is bound to rise. Even though the genetic causes of approximately one-third of PCD cases still remain to be found, the current knowledge can already be used to create new, accurate genetic tests for PCD that can accelerate the correct diagnosis and reduce the proportion of unexplained cases. This review aims to present the latest data on the relations between ciliary structure aberrations and their genetic basis.

Current genetic methodologies in the identification of disaster victims and in forensic analysis

This review presents the basic problems and currently available molecular techniques used for genetic profiling in disaster victim identification (DVI). The environmental conditions of a mass disaster often result in severe fragmentation, decomposition and intermixing of the remains of victims. In such cases, traditional identification based on the anthropological and physical characteristics of the victims is frequently inconclusive. This is the reason why DNA profiling became the gold standard for victim identification in mass-casualty incidents (MCIs) or any forensic cases where human remains are highly fragmented and/or degraded beyond recognition. The review provides general information about the sources of genetic material for DNA profiling, the genetic markers routinely used during genetic profiling (STR markers, mtDNA and single-nucleotide polymorphisms [SNP]) and the basic statistical approaches used in DNA-based disaster victim identification. Automated technological platforms that allow the simultaneous analysis of a multitude of genetic markers used in genetic identification (oligonucleotide microarray techniques and next-generation sequencing) are also presented. Forensic and population databases containing information on human variability, routinely used for statistical analyses, are discussed. The final part of this review is focused on recent developments, which offer particularly promising tools for forensic applications (mRNA analysis, transcriptome variation in individuals/populations and genetic profiling of specific cells separated from mixtures).

Longitudinal follow-up of exocrine pancreatic function in pancreatic sufficient cystic fibrosis patients using the fecal elastase-1 test.

BackgroundA progressive decline in pancreatic function is possible in cystic fibrosis (CF) patients with exocrine pancreatic sufficiency. The secretin–cholecystokinin test is invasive and not acceptable as a repeatable procedure for children. Steatorrhea, conversely, has low sensitivity. Therefore, the aim of the present study was to evaluate the usefulness of the noninvasive fecal elastase-1 (E1) test for the longitudinal assessment of exocrine pancreatic function (EPF) in pancreatic-sufficient (PS) CF patients. MethodsOne hundred eighty-four CF patients were included in the study. In all subjects, E1 concentrations and fecal fat excretion were measured. PS patients were followed for 5 years. ResultsAt the beginning of the study, 35 (19.0%) CF patients were PS, and 32 (17.4%) had normal E1 concentrations. Longitudinal measurements of E1 concentrations in PS patients with CF demonstrated stable enzyme output in 27 and gradual decrease in 8. The decrease was rapid in five infant patients and gradual in three older patients. The decrease of E1 concentrations preceded the appearance of steatorrhea in all eight subjects. ConclusionsThe decline of EPF in patients with CF appears more frequently during the first months and years of life. However, late PS to pancreatic-insufficient (PI) conversion is also possible. The appearance of maldigestion is preceded by the decrease of fecal E1 concentration. Thus, the fecal E1 test is a helpful screening tool for the longitudinal assessment of declining EPF in PS patients with CF to demonstrate pancreatic deterioration. In suspected patients, fecal fat excretion should be assessed.

T-cell acute lymphoblastic leukaemia: recent molecular biology findings

For many years, T‐cell acute lymphoblastic leukaemia (T‐ALL) has been considered and treated as a single malignancy, but divergent outcomes in T‐ALL patients receiving uniform treatment protocols encouraged intensive research on the molecular biology of this disease. Recent findings in the field demonstrate that T‐ALL is much more heterogeneous than originally believed and extremely diverse outcomes of patients require refinement of T‐ALL classification, leading to subtype‐specific adjustment of treatment. Many different biological features of T‐ALL blast cells have recently been found to contribute to disease development and patient outcome and their analysis could potentially be introduced into improved diagnostics and classification of the disease. This review focuses on five key issues of T‐ALL biology: chromosome aberrations, gene expression profiles, gene mutations, DNA methylation patterns, and immunoglobulin/T cell receptor (Ig/TCR) gene rearrangements. Additionally, molecular monitoring of minimal residual disease, by far the most reliable independent prognostic factor in T‐ALL, has been highlighted in the context of Ig/TCR gene rearrangements. Translation of this biological information into better prognostic classification and more effective treatment should lead to improvement of outcome in T‐ALL patients.

Analysis of exocrine pancreatic function in cystic fibrosis: one mild CFTR mutation does not exclude pancreatic insufficiency.

Background Cystic fibrosis (CF) is the most common cause of exocrine pancreatic insufficiency in childhood. The aim of the present study is to evaluate the correlation between genotype and exocrine pancreatic insufficiency in CF patients. The special emphasis was put on the analysis of mild CFTR mutations.

RPGR mutations might cause reduced orientation of respiratory cilia

RPGR gene encodes retinitis pigmentosa guanosine triphosphatase regulator protein, mutations of which cause 70% of the X‐linked retinitis pigmentosa (XLRP) cases. Rarely, RPGR mutations can also cause primary ciliary dyskinesia (PCD), a multisystem disorder characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis, and male subfertility. Two patients with PCD_RP and their relatives were analyzed using DNA sequencing, transmission electron microscopy (TEM), immunofluorescence (IF), photometry, and high‐speed videomicroscopy. The Polish patient carried a previously known c.154G>A substitution (p.Gly52Arg) in exon 2 (known to affect splicing); the mutation was co‐segregating with the XLRP symptoms in his family. The c.824 G>T mutation (p. Gly275Val) in the Australian patient was a de novo mutation. In both patients, TEM and IF did not reveal any changes in the respiratory cilia structure. However, following ciliogenesis in vitro, in contrast to the ciliary beat frequency, the ciliary beat coordination in the spheroids from the Polish proband and his relatives carrying the c.154G>A mutation was reduced. Analysis of the ciliary alignment indicated severely disturbed orientation of cilia. Therefore, we confirm that defects in the RPGR protein may contribute to syndromic PCD. Lack of ultrastructural defects in respiratory cilia of the probands, the reduced ciliary orientation and the decreased coordination of the ciliary bundles observed in the Polish patient suggested that the RPGR protein may play a role in the establishment of the proper respiratory cilia orientation. Pediatr Pulmonol. 2013; 48:352–363.

The EVI-1 gene — its role in pathogenesis of human leukemias

EVI-1 (ecotropic virus integration site-1) was at first identified as an integration site of the murine leukemia retrovirus in murine myeloid leukemias. It is involved in pathogenesis of mouse and human leukemias. EVI-1 expression may be activated by retroviral insertion or is caused by chromosomal translocations. EVI-1 gene is located on human chromosome 3, spans over 100 kb and contains 12 exons with ten coding exons. EVI-1 gene encodes 1051 amino acids DNA binding protein containing ten zinc finger repeats organized in two domains. The 145 kDa EVI-1 protein is localized in the nucleus. The structure of the EVI-1 protein indicates that it functions as a transcriptional factor of the zinc finger family. The role of this transcription factor in myeloid cell transformation and the target genes of EVI-1 is still unknown. Occurrence of a few EVI-1 fusion transcripts was shown. The role of this fusion proteins is still unclear. Mouse and human sequences of the gene show a high degree of homology; 91% in nucleotide sequence and 94% in amino acid sequence.