Mental health and the effects on methylation of stress-related genes in front-line versus other health care professionals during the second wave of COVID-19 pandemic: an Italian pilot study.

Healthcare workers experienced high degree of stress during COVID-19. Purpose of the present article is to compare mental health (depressive and Post-Traumatic-Stress-Disorders-PTSD-symptoms) and epigenetics aspects (degree of methylation of stress-related genes) in front-line healthcare professionals versus healthcare working in non-COVID-19 wards. Sixty-eight healthcare workers were included in the study: 39 were working in COVID-19 wards (cases) and 29 in non-COVID wards (controls). From all participants, demographic and clinical information were collected by an ad-hoc questionnaire. Depressive and PTSD symptoms were evaluated by the Patient Health Questionnaire-9 (PHQ-9) and the Impact of Event Scale-Revised (IES-R), respectively. Methylation analyses of 9 promoter/regulatory regions of genes known to be implicated in depression/PTSD (ADCYAP1, BDNF, CRHR1, DRD2, IGF2, LSD1/KDM1A, NR3C1, OXTR, SLC6A4) were performed on DNA from blood samples by the MassARRAY EpiTYPER platform, with MassCleave settings. Controls showed more frequent lifetime history of anxiety/depression with respect to cases (χ2 = 5.72, p = 0.03). On the contrary, cases versus controls presented higher PHQ-9 (t = 2.13, p = 0.04), PHQ-9 sleep item (t = 2.26, p = 0.03), IES-R total (t = 2.17, p = 0.03), IES-R intrusion (t = 2.46, p = 0.02), IES-R avoidance (t = 1.99, p = 0.05) mean total scores. Methylation levels at CRHR1, DRD2 and LSD1 genes was significantly higher in cases with respect to controls (p < 0.01, p = 0.03 and p = 0.03, respectively). Frontline health professionals experienced more negative effects on mental health during COVID-19 pandemic than non-frontline healthcare workers. Methylation levels were increased in genes regulating HPA axis (CRHR1) and dopamine neurotransmission (DRD2 and LSD1), thus supporting the involvement of these biological processes in depression/PTSD and indicating that methylation of these genes can be modulated by stress conditions, such as working as healthcare front-line during COVID-19 pandemic.

Genotype-phenotype correlation and functional studies in patients with cystic fibrosis bearing CFTR complex alleles.

BACKGROUND: The effect of complex alleles in cystic fibrosis (CF) is poorly defined for the lack of functional studies. OBJECTIVES: To describe the genotype-phenotype correlation and the results of either in vitro and ex vivo studies performed on nasal epithelial cells (NEC) in a cohort of patients with CF carrying cystic fibrosis transmembrane conductance regulator (CFTR) complex alleles. METHODS: We studied 70 homozygous, compound heterozygous or heterozygous for CFTR mutations: p.[Arg74Trp;Val201Met;Asp1270Asn], n=8; p.[Ile148Thr;Ile1023_Val1024del], n=5; p.[Arg117Leu;Leu997Phe], n=6; c.[1210-34TG[12];1210-12T[5];2930C>T], n=3; p.[Arg74Trp;Asp1270Asn], n=4; p.Asp1270Asn, n=2; p.Ile148Thr, n=6; p.Leu997Phe, n=36. In 39 patients, we analysed the CFTR gating activity on NEC in comparison with patients with CF (n=8) and carriers (n=4). Finally, we analysed in vitro the p.[Arg74Trp;Val201Met;Asp1270Asn] complex allele. RESULTS: The p.[Ile148Thr;Ile1023_Val1024del] caused severe CF in five compound heterozygous with a class I-II mutation. Their CFTR activity on NEC was comparable with patients with two class I-II mutations (mean 7.3% vs 6.9%). The p.[Arg74Trp;Asp1270Asn] and the p.Asp1270Asn have scarce functional effects, while p.[Arg74Trp;Val201Met;Asp1270Asn] caused mild CF in four of five subjects carrying a class I-II mutation in trans, or CFTR-related disorders (CFTR-RD) in three having in trans a class IV-V mutation. The p.[Arg74Trp;Val201Met;Asp1270Asn] causes significantly (p<0.001) higher CFTR activity compared with compound heterozygous for class I-II mutations. Furthermore, five of six compounds heterozygous with the p.[Arg117Leu;Leu997Phe] had mild CF, whereas the p.Leu997Phe, in trans with a class I-II CFTR mutation, caused CFTR-RD or a healthy status (CFTR activity: 21.3-36.9%). Finally, compounds heterozygous for the c.[1210-34TG[12];1210-12T[5];2930C>T] and a class I-II mutation had mild CF or CFTR-RD (gating activity: 18.5-19.0%). CONCLUSIONS: The effect of complex alleles partially depends on the mutation in trans. Although larger studies are necessary, the CFTR activity on NEC is a rapid contributory tool to classify patients with CFTR dysfunction.

Clinical expression of cystic fibrosis in a large cohort of Italian siblings.

BACKGROUND: A clinical heterogeneity was reported in patients with Cystic Fibrosis (CF) with the same CFTR genotype and between siblings with CF. METHODS: We investigated all clinical aspects in a cohort of 101 pairs of siblings with CF (including 6 triplets) followed since diagnosis. RESULTS: Severe lung disease had a 22.2% concordance in sib-pairs, occurred early and the FEV1% at 12 years was predictive of the severity of lung disease in the adulthood. Similarly, CF liver disease occurred early (median: 15 years) and showed a concordance of 27.8% in sib-pairs suggesting a scarce contribution of genetic factors; in fact, only 2/15 patients with liver disease in discordant sib-pairs had a deficiency of alpha-1-antitrypsin (a known modifier gene of CF liver phenotype). CF related diabetes was found in 22 pairs (in 6 in both the siblings). It occurred later (median: 32.5 years) and is strongly associated with liver disease. Colonization by P. aeruginosa and nasal polyposis that required surgery had a concordance > 50% in sib-pairs and were poorly correlated to other clinical parameters. The pancreatic status was highly concordant in pairs of siblings (i.e., 95.1%) but a different pancreatic status was observed in patients with the same CFTR mutations. This suggests a close relationship of the pancreatic status with the "whole" CFTR genotype, including mutations in regulatory regions that may modulate the levels of CFTR expression. Finally, a severe course of CF was evident in a number of patients with pancreatic sufficiency. CONCLUSIONS: Physicians involved in care of patients with CF and in genetic counseling must be aware of the clinical heterogeneity of CF even in sib-pairs that, at the state of the art, is difficult to explain.

Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events.

Classic Ehlers-Danlos syndrome (cEDS) is characterized by skin hyperelasticity, joint hypermobility, increased tendency to bruise, and abnormal scarring. Mutations in type V collagen, a regulator of type I collagen fibrillogenesis, underlie this type of EDS. In this article we report a genetic and clinical analysis of an Italian family that carried missense mutation c.934 C>T (p.R312C) in the COL1A1 gene. Literature review showed an association between this missense mutation and vascular complications. Genetic screening conducted on Italian family members, revealed that vascular events are absent. In conclusion, genetic and clinical data confirm the extreme heterogeneity of EDS. Nevertheless, vascular events could be a risk factor and periodical clinical evaluation could be relevant.

RETRACTED: Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events.

The article entitled "Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events" has been retracted because the description and characterization of the disease in a family may have been previously published. Upon publication of this article we were notified by an author of a study appearing in 2016 in another journal claiming that characteristics and symptoms of the family described closely matched their study, and that the two studies describe the same family. Whereas constituent family members appearing in both articles were not identical (differing by one member), symptoms and diagnoses of each family proband appeared to be consistent in both studies, leading to the editors' conclusion that it is likely that the same family was being described in two separate articles.The corresponding author of the article in Dermatology Online Journal was informed of this incident, and responded with the assertion that they were unaware of the study published in 2016, and provided no additional information. They further requested that their article be retracted. In light of the available information and author's request, the editors of Dermatology Online Journal have retracted this article.The original article was published on July15, 2018 and corrected on September 15, 2018.The original article was published on July15, 2018 and corrected on September 15, 2018.

Survival and dementia in GBA-associated Parkinson's disease: The mutation matters.

OBJECTIVE: The objective of this work was to investigate survival, dementia, and genotype-phenotype correlations in patients with Parkinson's disease (PD) with and without mutations on the glucocerebrosidase gene (GBA). METHODS: We included 2,764 unrelated consecutive PD patients: 123 GBA carriers (67 mild-p.N370S and 56 severe mainly p.L444P) and 2,641 noncarriers. Brain perfusion and dopamine transporter imaging was analyzed, including dementia with Lewy Bodies (DLB) as an additional control group. RESULTS: Multivariable analysis adjusted by sex, age at onset, and disease duration attributed to GBA carriers a greater risk for dementia (hazard ratio [HR] = 3.16; p < 0.001) and death (HR = 1.85; p = 0.002) than noncarriers. When dementia was introduced in the model as a time-dependent covariate, the mortality risk remained greater in carriers (HR = 1.65; p = 0.016), suggesting that other clinical features are likely to contribute to reduced survival. At last examination, GBA carriers had worse motor symptoms, particularly nondopaminergic features. Carriers of severe mutations had greater risk for dementia compared to mild mutations (p < 0.001), but similar mortality risk. Consistent with clinical data, GBA carriers showed reduced posterior parietal and occipital cortical synaptic activity and nigrostriatal function than PD noncarriers. Neuroimaging features of carriers of mild mutations overlapped with PD noncarriers, whereas carriers of severe mutations were closer to DLB. INTERPRETATION: Survival is reduced in GBA carriers compared to noncarriers; this seems to be partially independent from the increased risk for early dementia. The risk for dementia is strongly modulated by type of mutation. In the clinical continuum between PD and DLB, patients with GBA mutations seem to localize midway, with carriers of severe mutations closer to DLB than to idiopathic PD. Ann Neurol 2016;80:662-673.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma.

BACKGROUND: Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. METHODS: Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing ß-thalassaemia and 7 cystic fibrosis. RESULTS: In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian ß-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. CONCLUSIONS: COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed.

Hb Mozhaisk [ß92(F8)His→Arg; HBB: c.278A>G] as a De Novo Mutation in a Child of Mixed Ethnic Origins.

Approximately 150 variants described in the HbVar database have been found to be unstable and about 80.0% of these are on the ß-globin gene. We describe the case of a 3-year-old child who presented at the emergency room with fever and asthenia. Hematological data suggested severe hemolytic anemia. Sequencing of the ß-globin gene revealed the mutation HBB: c.278A>G at codon 92 in a heterozygous state, reported as Hb Mozhaisk in the HbVar database. Other family members did not have Hb Mozhaisk, thus, this variant is due to a de novo mutation. Because of the rarity of this globin variant, we believe it is important to report similar cases, to have a more complete phenotype description of the pathology and define an adequate reproductive risk for couples, considering the dominant inheritance pattern (hence an inheritance risk of 50.0%).

Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis.

Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG[11-12]T5 allele-known to be associated with decreased CFTR mRNA levels-the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

Genotypes and phenotypes in cystic fibrosis and cystic fibrosis transmembrane regulator-related disorders.

Cystic fibrosis (CF) is characterized by remarkable variability in severity, rate of disease progression, and organ involvement. In spite of the considerable amount of data collected on the relationship between genotype and phenotype in CF, this is still a challenging matter of debate. Barriers to the interpretation of this connection are the large number of mutations in the CF transmembrane regulator (CFTR) gene, the difficulties in attributing several of them to a specific mode of dysfunction, and a limited number of the almost 2,000 mutations so far detected, which have been clinically annotated. In addition to that, the heterogeneity of clinical manifestations in individuals with the same CFTR genotypes indicates that disease severity is modulated by other genes and by environmental factors, of which the most relevant is possibly treatment in its aspects of appropriateness, early start in life, and adherence. The phenotype variability extends to conditions, named CFTR-related disorders, which are connected with CFTR dysfunction, but do not satisfy diagnostic criteria for CF. The current level of knowledge does not allow use of the CFTR genotype to predict individual outcome and cannot be used as an indicator of CF prognosis. This might change with the development of treatments targeting specific mutations and possibly capable of changing the natural history of the disease.

Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.

Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene.

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.

Amniotic mesenchymal stem cells: a new source for hepatocyte-like cells and induction of CFTR expression by coculture with cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33-50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

VPS13C-associated Parkinson's disease: Two novel cases and review of the literature.

VPS13C is a protein-coding gene involved in the regulation of mitochondrial function through the endolysosomal pathway in neurons. Homozygous and compound heterozygous VPS13C mutations are etiologically associated with early-onset Parkinson's disease (PD). Moreover, recent studies linked biallelic VPS13C mutations with the development of dementia with Lewy bodies (DLB). Neuropathological studies on two mutated subjects showed diffuse Lewy body disease. In this article, we report the clinical and genetic findings of two subjects affected by early-onset PD carrying three novel VPS13C mutations (i.e., one homozygous and one compound heterozygous), and review the previous literature on the genetic and clinical findings of VPS13C-mutated patients, contributing to the knowledge of this rare genetic alpha-synucleinopathy.

An interactive computer program can effectively educate potential users of cystic fibrosis carrier tests.

The demand for cystic fibrosis (CF) carrier testing is steadily growing, not only from individuals with raised a priori carrier risk, but also from the general population. This trend will likely exceed the availability of genetic counselors, making it impossible to provide standard face-to-face genetic counseling to all those asking for the test. In order to reduce the time needed to educate individuals on the basics of the disease, its genetic transmission, and carrier testing peculiarities, we developed an educational method based on an interactive computer program (IC). To assess the effectiveness of this program and to compare it to a classical genetic counseling session, we conducted a comparative trial. In a population setting of people undergoing assisted reproduction, 44 individuals were randomly assigned to either receiving standard one-on-one genetic counseling or education by the IC program. We measured pre- and post-intervention knowledge about CF genetic transmission and carrier testing. Starting from an equivalent baseline of correct answers to a specially designed multiple-choice questionnaire (47% in the counselor group and 45% in the computer group) both groups showed a highly significant and similar increase (reaching 84% in the counselor group and 85% in the computer group). The computer program under evaluation can successfully educate individuals considering genetic testing for CF.

A polymorphism in the 5' UTR of the DEFB1 gene is associated with the lung phenotype in F508del homozygous Italian cystic fibrosis patients.

BACKGROUND: The identification of cystic fibrosis (CF) patients who are at greater risk of lung damage could be clinically valuable. Thus, we attempted to replicate previous findings and verify the possible association between three single nucleotide polymorphisms (SNPs c.-52G>A, c.-44C>G and c.-20G>A) in the 5' untranslated region (5' UTR) of the ß defensin 1 (DEFB1) gene and the CF pulmonary phenotype. METHODS: Genomic DNA from 92 Italian CF patients enrolled in different regional CF centres was extracted from peripheral blood and genotyped for DEFB1 SNPs using TaqMan(®) allele specific probes. In order to avoid genetic confounding causes that can account for CF phenotype variability, all patients were homozygous for the F508del CFTR mutation, and were then classified on the basis of clinical and functional data as mild lung phenotype (Mp, n=50) or severe lung phenotype patients (Sp, n=42). RESULTS: For the c.-20G>A SNP, the frequency of the A allele, as well as the AA genotype, were significantly more frequent in Mp than in Sp patients, and thus this was associated with a protective effect against severe pulmonary disease (OR=0.48 and 0.28, respectively). The effect of the c.-20G>A A allele is consistent with a recessive model, and the protective effect against Sp is exerted only when it is present in homozygosis. For the other two SNPs, no differences were observed as allelic and genotypic frequency in the two subgroups of CF patients. CONCLUSIONS: Our results, although necessary to be confirmed in larger and multiethnic populations, reinforce DEFB1 as a candidate modifier gene of the CF pulmonary phenotype.

Use of parthenogenetic activation of human oocytes as an experimental model for evaluation of polar body based PGD assay performance.

PURPOSE: To develop an experimental model to assess the feasibility of polar body preimplantation genetic diagnosis without requiring oocyte fertilization. METHODS: First polar body was removed from donated oocytes and second polar body was biopsied after parthenogenetic activation. Molecular analysis on both polar bodies involved a fluorescent multiplex polymerase chain reaction of short-tandem repeat markers, closely linked to genes of interest. Main outcome measures were: allele segregation through polar bodies and haploid nucleus, recombination rates between alleles and frequency of Allele Drop Out. RESULTS: Twenty-six out of 39 oocytes extruded a second polar body after activation. Ninety-two percent of the first polar bodies and 20 out of 26 (77%) second polar bodies were successfully amplified. Eighty percent of first polar bodies were heterozygous for CFTR and 55% for HBB. Analysis of second polar bodies predicted the genotype of the oocytes in case of heterozygous first polar body, and validated results in homozygous cases. Frequency of allele drop out was 4%. CONCLUSIONS: Our model confirms that polar body preimplantation genetic diagnosis for single gene disorders can be evaluated using parthenogenetic oocytes and offers an option to set up procedures without requiring oocyte fertilization.

Risk of Atypical HUS Among Family Members of Patients Carrying Complement Regulatory Gene Abnormality.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is mainly due to complement regulatory gene abnormalities with a dominant pattern but incomplete penetrance. Thus, healthy carriers can be identified in any family of aHUS patients, but it is unpredictable if they will eventually develop aHUS. METHODS: Patients are screened for 10 complement regulatory gene abnormalities and once a genetic alteration is identified, the search is extended to at-risk family members. The present cohort study includes 257 subjects from 71 families: 99 aHUS patients (71 index cases + 28 affected family members) and 158 healthy relatives with a documented complement gene abnormality. RESULTS: Fourteen families (19.7%) experienced multiple cases. Over a cumulative observation period of 7595 person-years, only 28 family members carrying gene mutations experienced aHUS (overall penetrance of 20%), leading to a disease rate of 3.69 events for 1000 person-years. The disease rate was 7.47 per 1000 person-years among siblings, 6.29 among offspring, 2.01 among parents, 1.84 among carriers of variants of uncertain significance, and 4.43 among carriers of causative variants. CONCLUSIONS: The penetrance of aHUS seems a lot lower than previously reported. Moreover, the disease risk is higher in carriers of causative variants and is not equally distributed among generations: siblings and the offspring of patients have a much greater disease risk than parents. However, risk calculation may depend on variant classification that could change over time.

Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.