Truncating mutations in exons 20 and 21 of OFD1 can cause primary ciliary dyskinesia without associated syndromic symptoms.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a motile ciliopathy, whose symptoms include airway infections, male infertility and situs inversus. Apart from the typical forms of PCD, rare syndromic PCD forms exist. Mutations of the X-linked OFD1 gene cause several syndromic ciliopathies, including oral-facial-digital syndrome type 1, Joubert syndrome type 10 (JBTS10), and Simpson-Golabi-Behmel syndrome type 2, the latter causing the X-linked syndromic form of PCD. Neurological and skeletal symptoms are characteristic for these syndromes, with their severity depending on the location of the mutation within the gene. OBJECTIVES: To elucidate the role of motile cilia defects in the respiratory phenotype of PCD patients with C-terminal OFD1 mutations. METHODS: Whole-exome sequencing in a group of 120 Polish PCD patients, mutation screening of the OFD1 coding sequence, analysis of motile cilia, and magnetic resonance brain imaging. RESULTS: Four novel hemizygous OFD1 mutations, in exons 20 and 21, were found in men with a typical PCD presentation but without severe neurological, skeletal or renal symptoms characteristic for other OFD1-related syndromes. Magnetic resonance brain imaging in two patients did not show a molar tooth sign typical for JBTS10. Cilia in the respiratory epithelium were sparse, unusually long and displayed a defective motility pattern. CONCLUSION: Consistent with the literature, truncations of the C-terminal part of OFD1 (exons 16-22) almost invariably cause a respiratory phenotype (due to motile cilia defects) while their impact on the primary cilia function is limited. We suggest that exons 20-21 should be included in the panel for regular mutation screening in PCD.

CFAP300: Mutations in Slavic Patients with Primary Ciliary Dyskinesia and a Role in Ciliary Dynein Arms Trafficking.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous hereditary disease from a class of ciliopathies. In spite of the recent progress, the genetic basis of PCD in one-third of patients remains unknown. In search for new genes and/or mutations, whole-exome sequencing was performed in 120 unrelated Polish patients with PCD, in whom no genetic cause of PCD was earlier identified. Among a number of pathogenic variants in PCD genes, mutations in CFAP300 (alias C11orf70) were detected. Extended screening in the whole Polish PCD cohort revealed the relatively high frequency (3.6%) of otherwise rare c.[198_200 del_insCC] variant, indicating that it should be included in population-specific genetic tests for PCD in Slavic populations. Immunofluorescence analysis of the respiratory epithelial cells from patients with CFAP300 mutations revealed the absence or aberrant localization of outer and inner dynein arm markers, consistent with transmission electron microscope images indicating the lack of both dynein arms. Interestingly, the disparate localization of DNAH5 and DNALI1 proteins in patients with CFAP300 mutations suggested differential mechanisms for the trafficking of preassembled outer and inner dynein arms to the axoneme. The profile of CFAP300 expression during ciliogenesis in suspension culture was consistent with its role in cilia assembly. Gene silencing experiments, performed in a model organism, Schmidtea mediterranea (flatworm), pointed to the conserved role of CFAP300 in ciliary function.

ZMYND10--Mutation Analysis in Slavic Patients with Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare recessive disease with a prevalence of 1/10,000; its symptoms are caused by a kinetic dysfunction of motile cilia in the respiratory epithelium, flagella in spermatozoids, and primary cilia in the embryonic node. PCD is genetically heterogeneous: genotyping the already known PCD-related genes explains the genetic basis in 60-65% of the cases, depending on the population. While identification of new genes involved in PCD pathogenesis remains crucial, the search for new, population-specific mutations causative for PCD is equally important. The Slavs remain far less characterized in this respect compared to West European populations, which significantly limits diagnostic capability. The main goal of this study was to characterize the profile of causative genetic defects in one of the PCD-causing genes, ZMYND10, in the cohort of PCD patients of Slavic origin. The study was carried out using biological material from 172 unrelated PCD individuals of Polish origin, with no causative mutation found in nine major PCD genes. While none of the previously described mutations was found using the HRM-based screening, a novel frameshift mutation (c.367delC) in ZMYND10, unique for Slavic PCD population, was found in homozygous state in two unrelated PCD patients. Immunofluorescence analysis confirmed the absence of outer and inner dynein arms from the ciliary axoneme, consistent with the already published ZMYND10-mutated phenotype; cDNA analysis revealed the lack of ZMYND10 mRNA, indicating nonsense-mediated decay of the truncated transcript.

Effects of age and gender on micronucleus and chromosome nondisjunction frequencies in centenarians and younger subjects.

Studies have shown a significant increase in chromosome aneuploidy with age. The aim of this study was to elucidate whether the age-related changes in the level of hypoploidy correlate with the occurrence of micronuclei (MN) and chromosome nondisjunction (ND) in men and women. We analyzed cytokinesis-blocked (binucleated) lymphocytes treated with cytochalasin B, from 127 donors varying in gender and age including 53 centenarians. Fluorescent in situ hybridization with probes specific for several autosomes (1, 4, 6, 8, 20) and for the sex chromosomes was applied to analyze the chromosomal content of MN and to analyze the frequency of reciprocal loss and gain due to ND in binucleated interphase cells. The general level of MN in Giemsa-stained preparations was higher in women and in both genders increased with age until approximately 70 years and ranged, depending on age group, from 0.5 to 1.4% in men and from 0.9 to 1.8% in women. Gender-related differences were mostly observed in the younger age groups (< or =50 years), with an almost two-fold difference between men and women (P < 0.005). Frequencies of autosome-positive MN in both genders and of sex chromosome-positive MN in men were comparable and remained unchanged in older groups. The frequency of X-positive MN in women was higher than the average frequency of autosome-positive MN and continued to increase until the oldest age. The frequency of NDs involving the analyzed chromosomes was on average two-fold higher in women than in men. In both genders, the frequency of NDs increased with age and was, on average, an order of magnitude higher than that of cells with MN, consistent with the previous reports that the efficiency of elimination of micronucleated cells is higher than of the cells presenting chromosome ND.

Correlation between the level of cytogenetic aberrations in cultured human lymphocytes and the age and gender of donors.

To answer whether the age-related accumulation of chromosomal damage differs in men and women, and whether the aberration level in centenarians is proportional to their age, cytogenetic aberrations in dividing cells were analyzed. G-band karyotyping of mitotic spreads from lymphocytes was performed in 52 Polish centenarians and 71 controls (aged 21-78). Statistical evaluation was performed using nonparametric tests and regression analysis. The average level of all chromosomal aberrations was comparable in centenarians of both genders, but the age-related increase in chromosomal damage occurred faster in women than in men. Aging in both genders was marked by the increasing level of all aberrations rather than by chromosome-specific changes; the loss of X chromosome was the leading contributor in women. The age-related increase in the level of chromosomal damage reflected accumulation of dividing cells with a small number of aberrations. Individuals who survive to the extreme old age appear to accumulate aberrations at the slower rate.

Manifestations of ageing at the cytogenetic level.

The effects of ageing in humans appear to be a combination of influence of genetically programmed phenomena and exogenous environmental factors, and take place at the cellular level (senescence), rather than at the level of the organism. There are many processes, which occur in somatic cells as a consequence of DNA replication (accumulation of DNA errors or mutations that outstrip repair processes, telomere shortening, deregulation of apoptosis, etc.) and which drive replicative senescence in human cells. DNA errors are considered to be critical primary lesions in the formation of chromosomal aberrations. It can be concluded that the chromosome aberrations are biomarkers of ageing in human cells. Studies of human metaphases, interphase nuclei and micronuclei showed the increase in loss of chromosomes and the increase in frequency of stable chromosome aberrations as a function of age.

The 102-year old woman with translocation (7;12) and infertility in anamnesis.

In this study we present a 102-year old woman carrying a (7;12)(q11.3;q14) translocation. A woman displays a normal phenotype and infertility in anamnesis. This is the first report linking t(7;12) and infertility.

Infertility status of male individuals with abnormal spermiogram evaluated by cytogenetic analysis and in vitro sperm penetration assay.

BACKGROUND: The possibility of a link between altered sperm morphology and functional ability, possibly reflected on the genetic level, is still a matter of controversy. MATERIAL/METHODS: 60 infertile males with pathological spermiogram and 14 healthy individuals with confirmed in vivo fertility were selected for our study. Ejaculated sperm was assessed according to the standard criteria set by the World Health Organization, which constituted a basis for subgroup classification. The sperm penetration assay (SPA) using human sperm and hamster oocytes was subsequently performed with concomitant cytogenetic analysis of peripheral blood leukocytes (PBL). RESULTS: More than 55% of infertile males with pathological spermiogram exhibited a decreased ability for penetration of the oocyte (SPAL30%). The lowest penetration ability was observed in the semen of patients with oligoasthenoteratozoospermia (OAT), of whom as many as 75% presented negative SPA values. We noted only 1 case of chromosome aberration (Robertsonian translocation) in the analyzed cohort of infertile patients. Strikingly, in the identified group of infertile patients with chromosome structural polymorphism (acrocentric chromosomes), we revealed a statistically significant decreased rate of sperm - oocyte penetration. CONCLUSIONS: A causative link may exist between sperm malfunction and a specific feature of the chromosome variants detected in the blood leukocytes of infertile males with pathological spermiogram.