Huda Mussaffi

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.

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

Evidence of Intestinal Inflammation in Patients With Cystic Fibrosis

Objectives: Treatment with pancreatic enzymes fails to completely correct malabsorption and gastrointestinal symptoms in patients with cystic fibrosis (CF). The aim of the present study was to examine the small intestine of patients with CF without overt evidence of gastrointestinal disease using capsule endoscopy (CE). Methods: Patients with CF received the agile patency capsule and, depending on the result of that procedure, then underwent standard CE using the PillCam SB capsule (Given Imaging, Yokneam, Israel). A stool specimen was taken on the same day as the CE for determination of the calprotectin level. Results: Forty-two patients with CF ages 10 to 36 years were included; 29 had pancreatic insufficiency. One patient failed to excrete the patency capsule after 36 hours and was withdrawn from the study. Pulmonary function was mild to moderate with FEV1 68.5% ± 16% predicted. Review of the CE videos showed that most of the patients had varying degrees of diffuse areas of inflammatory findings in the small bowel including edema, erythema, mucosal breaks, and frank ulcerations. There were no adverse events. Fecal calprotectin levels were markedly high in patients with pancreatic insufficiency, 258 μg/g (normal <50). Conclusions: Small bowel mucosal pathology may be detected using CE in most of the patients with CF. The high fecal calprotectin levels found are suggestive of mucosal inflammation, which may correlate with the CE findings. Additional study is required to examine the possible relation of these mucosal lesions, which may be part of a newly identified enteropathy associated with CF, with persistent intestinal malabsorption in many of these patients.

Nontuberculous mycobacteria in cystic fibrosis associated with allergic bronchopulmonary aspergillosis and steroid therapy

Nontuberculous mycobacterial (NTM) infection, particularly due to Mycobacterium abscessus, is an emerging disease that can be relentlessly progressive, particularly in cystic fibrosis (CF) patients. The risk factors that were associated with this increasingly symptomatic infection in a group of CF patients were investigated. A total of 139 CF patients aged 2–52 yrs were reviewed. Sputum was cultured for NTM annually or whenever clinical deterioration was unexplained. In total, 12 patients (8.6%) had positive cultures and six (4.3%) met the criteria for NTM pulmonary disease (five with M. abscessus). Five had allergic bronchopulmonary aspergillosis (ABPA) compared with one out of 133 patients without NTM disease. Five had received systemic steroids (four as a treatment for ABPA) compared with only one out of 133 without NTM lung disease. All six NTM patients deteriorated markedly following mycobacterial infection, and forced expiratory volume in one second dropped 18–46%. Despite prolonged triple antibiotic therapy, M. abscessus was not eradicated, and four out of six did not return to baseline clinically. In conclusion, severe nontuberculous mycobacterial lung disease, particularly with Mycobacterium abscessus, is becoming a perplexing challenge in cystic fibrosis patients. Allergic bronchopulmonary aspergillosis and systemic steroids appear to be risk factors, although small patient numbers limit this to a descriptive observation. When pulmonary condition deteriorates, increased surveillance for mycobacteria would enable prompt diagnosis and treatment.

Increased bronchial responsiveness to exercise and histamine after allergen challenge in children with asthma

Nonspecific bronchial reactivity to exercise and histamine was measured in nine children with asthma before and within nine days after allergen inhalation. All patients developed an immediate fall in FEV1 of greater than or equal to 16% after allergen inhalation, and five children also developed a definite late asthmatic response with a fall in peak expiratory flow of greater than or equal to 14% after 3 to 8 hours. Mean postexercise fall in FEV1 (delta FEV1) of the whole group was significantly greater after allergen challenge compared with that of control subjects (29 +/- 6% and 16 +/- 4%; p = 0.013). There was no change in refractoriness to repeated exercise after allergen challenge. The mean provocation concentration of histamine causing a decrease in FEV1 of 20% of the whole group was less after allergen challenge compared with that of control subjects (0.47 +/- 0.18 and 0.62 +/- 0.13), but this was not statistically significant (p = 0.19). Of the five children with late allergen reactions, all demonstrated increased histamine sensitivity, and all four children who developed definite exercise-induced asthma also demonstrated increased sensitivity to exercise. Of the four children without late allergen reactions, none demonstrated increase histamine sensitivity, but two of the three children with definite exercise-induced asthma demonstrated increased sensitivity to exercise. It may be that sensitivity to exercise is more easily affected by nonspecific reactivity than sensitivity to histamine. It is concluded that increased bronchial responsiveness to both exercise and histamine occur after allergen provocation in patients with asthma.

Encouraging Pulmonary Outcome for Surviving, Neurologically Intact, Extremely Premature Infants in the Postsurfactant Era

OBJECTIVE The aim of this study was to determine the long-term pulmonary outcome of extreme prematurity at a single tertiary-care center from 1997 to 2001 in the postsurfactant era. METHODS We assessed symptoms, exhaled nitric oxide, spirometry, methacholine challenge (provocative concentration of methacholine required to decrease FEV₁ by 20% [PC(20)]), lung volumes, diffusion, and cardiopulmonary exercise tolerance. RESULTS Of 279 infants born, 192 survived to discharge, and 79 of these developed bronchopulmonary dysplasia (BPD) (65 mild, 12 moderate, two severe). We studied a subgroup of 53 neurologically intact preterm subjects aged 10 ± 1.5 years (28 with BPD [born, 26.2 ± 1.4 weeks; birth weight, 821 ± 164 g] and 25 without BPD [born, 27.2 ± 1 weeks; birth weight, 1,050 ± 181 g]) and compared them with 23 term control subjects. Of the BPD cases, 21 were mild, seven were moderate, and none was severe; 77.4% of subjects received antenatal steroids, and 83% received postnatal surfactant. Sixty percent of the preterm subjects wheezed at age < 2 years compared with 13% of the control subjects (P < .001), but only 13% wheezed in the past year compared with 0% of control subjects (not significant). For preterm and control subjects, respectively (mean ± SD), FEV₁ % predicted was 85% ± 10% and 94% ± 10% (P < .001), with limited reversibility; residual volume/total lung capacity was 29.3% ± 5.5% and 25% ± 8% (P < .05); diffusing capacity/alveolar volume was 89.6% ± 9.2% and 97% ± 10% (P < .005); and PC(20) was 6.5 ± 5.8 mg/mL and 11.7 ± 5.5 mg/mL (P < .001). PC(20) was < 4 mg/mL in 49% of preterm subjects despite normal exhaled nitric oxide. Most measurements were similar in premature subjects with and without BPD. Peak oxygen consumption and breathing reserve were normal, but % predicted maximal load (measured in Watts) was 69% ± 15% for subjects with BPD compared with 88% ± 23% for subjects without and 86% ± 20% for control subjects (P < .01). CONCLUSIONS Pulmonary outcome was encouraging at mid-childhood for neurologically intact survivors in the postsurfactant era. Despite mechanical ventilation and oxygen therapy, most had no or mild BPD. Changes found probably reflect the hypoplastic lungs of prematurity.

Biological function of the soluble CEACAM1 protein and implications in TAP2-deficient patients.

Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory signals controlling NK killing activity. It is therefore surprising to learn that TAP2‐deficientpatients suffer from autoimmune manifestations only occasionally in later stages of life. We have previously described that the CEACAM1‐mediated inhibitory mechanism of NK cytotoxicity plays a major role in controlling NK autoreactivity in three newly identified TAP2‐deficient siblings. This novel mechanism probably compensates for the lack of MHC class I‐mediated inhibition. The CEACAM1 protein can also be present in a soluble form and the biological function of the soluble form of CEACAM1 with regard to NK cells has not been investigated. Here we show that the homophilic CEACAM1 interactions are abrogated in the presence of soluble CEACAM1 protein in a dose‐dependent manner. Importantly, the amounts of soluble CEACAM1 protein detected in sera derived from the TAP2‐deficient patients were dramatically reduced as compared to healthy controls. This dramatic reduction does not depend on the membrane‐bound metalloproteinase activity. Thus, the expression of CEACAM1 and the absence of soluble CEACAM1 observed in the TAP2‐deficient patients practically maximize the inhibitory effect and probably help to minimize autoimmunity in these patients.

DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

Urogenital abnormalities in male children with cystic fibrosis.

Background: Congenital bilateral absence of the vas deferens (CBAVD) is presumed to occur prenatally and is present in over 99% of adult males with cystic fibrosis (CF). Aims: To describe ultrasonic features in male children with CF. We aimed to describe urogenital anomalies, comparing pancreatic sufficient and insufficient CF patients. Methods: Pelvic and scrotal ultrasonography were performed in 12 boys with CF aged 2–12 years and 16 age matched healthy controls. Results: Nine patients had pancreatic insufficiency (PI): seven had two severe mutations and two had unknown mutations. Three boys were pancreatic sufficient (PS), two with splicing mutations (5T and 3849+10kb C–T respectively) and borderline sweat tests. Seminal vesicles were visualised in 5/12 patients and 8/16 controls, compared to non-visualisation reported in all adults with CBAVD. Testicular microlithiasis was found in 4/18 PI, 0/6 PS, and 0/32 control testes, compared to 0.6–1.4% in healthy males and 15% in CF adults; 7/18 PI, 4/6 PS, and 0/32 control testes were smaller than predicted for age. The epididymal head was non-homogeneous with cysts, hypo-, or hyper-echogenicity in 5/18 PI, 1/6 PS, and 0/32 control testes. Conclusions: Genital abnormalities may occur early in CF, but are less common than described in adults. They are found more often in pancreatic insufficient than in pancreatic sufficient CF patients. However, a positive finding, if present, may aid in the diagnosis of the latter. A larger longitudinal study is recommended to better define the onset and progression of urogenital abnormalities.

Hemoptysis in Israeli CF patients — Prevalence, treatment, and clinical characteristics

OBJECTIVE To identify the characteristics of CF patients with hemoptysis in Israel and to compare clinical features and risk factors to a control group of CF patients without hemoptysis. DESIGN Retrospective chart review. PATIENTS All CF patients in Israel who experienced hemoptysis between 2001 and 2005 and a control group of sex- and age-matched patients with no history of hemoptysis. RESULTS 40/440 CF patients (9.1%) experienced hemoptysis during the study period, 50% were male. Ten patients (25%) were under 13 years old at the first hemoptysis episode. Pulmonary exacerbation was the precipitating factor in 90%. Twenty three patients showed moderate or major hemoptysis. 35/40 patients responded well to conservative therapy. Bronchial artery embolization (BAE) was performed in 5 patients with no recurrence of bleed within 24 h. However all of these patients experienced recurrent hemoptysis. One patient died during the follow-up period because of end stage lung disease. Pulmonary function tests, body-mass index, coagulation tests, pancreatic status, presence of bronchiectasis, sputum cultures and genetic mutations were similar in the two groups. A high incidence (57.5%) of associated diseases including cystic fibrosis related diabetes, cirrhosis and portal hypertension, and distal intestinal obstruction syndrome was found among hemoptysis patients, compared to only 5.2% in the control group (p<0.001). CONCLUSIONS Hemoptysis, even major, did not seem to be a risk factor for mortality in our patients. A higher incidence of hemoptysis was found in our pediatric patients compared to other series. BAE shows a high immediate rate of success in controlling hemoptysis, but does not prevent future disease.