3849+10 kb C→T mutation and disease severity in cystic fibrosis

lesions in Macaca fascicularis as revealed by positron emission tomography and magnetic resonance imaging. Arch Toxicol 1992; 66: 403-07. 21 Brouillet EP, Shinobu L, McGarvey U, Hochberg F, Beal MF. Manganese injection into rat striatum produces excitotoxic lesions impairing energy metabolism. Exp Neurol 1993; 120: 89-94.

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E, Kaminsky Y, Grau E, et al. Brain ATP depletion induced ammonia intoxication in rats is mediated by activation of the NMDA receptor and Na-K-ATPase. J Neurochem 1994; 63: 2172-78. 25 Mousseau DD, Perney P, Layrargues GP, Butterworth RF. Selective loss of pallidal dopamine D2 receptor density in hepatic encephalopathy. Neurosci Lett 1993; 162: 192-96. 26 Rao VL, Giguere JF, Layrargues GP, Butterworth RF. Increased activities of MAOA and MAOB in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Brain Res 1993; 621: 349-52. 27 Lai JC, Chan AW, Leung TK, Minski MJ, Lim L. Neurochemical changes in rats chronically treated with a high concentration of manganese chloride. Neuochem Res 1992; 17: 841-47. 24 Kosenko

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3849+10 kb C→T mutation and disease

severity in cystic fibrosis

Summary

Introduction

50% of patients with cystic fibrosis (CF) are homozygous for the &Dgr;F508 mutation, but the remainder have at least one of many other less common mutations. The 3849+10 kb C→T splice mutation seems to be associated with less severe disease. We report ten CF patients who are hemizygous for this mutation. Three male patients do not have azoospermia (sperm counts 12, 53, and 198×106/mL). Another boy died before CF was diagnosed; his genital tract appeared normal at necropsy. All patients had clinically sufficient exocrine pancreatic function. Pulmonary disease was delayed in onset in most of these patients (range <1 to 16; median 9 years), but then became severe in some, progressing to death (in two) and need for transplantation in one. Sweat chloride concentration was abnormal (80 mmol/L) in one patient but in others was in the intermediate range (45-65 mmol/L). There was substantial variation in pulmonary disease severity within sibships. These observations suggest some dissociation in genetically determined severity between different organs. There was some evidence among these patients of a tendency for disease of the genital tract to become progressively more severe. Perhaps sperm banking should be offered to male patients. We speculate that some normal CF transmembrane conductance regulator is made in patients with this splice abnormality.

Cystic fibrosis (CF) is characterised by high sweat chloride concentrations, progressive pulmonary disease, and exocrine pancreatic insufficiency. The autosomal recessive gene has been identified and cloned.’ Although about 50% of patients are homozygous for the AF508 mutation, more than 350 other mutations are known; some are associated with milder disease, such as preserved exocrine pancreatic function.3 More than 96% of men who are homozygous for AF508 are azoospermic ;4 this defect is thought to be due to prenatal anatomical interruption of the vas deferens. We report ten patients who are hemizygous for a splice mutation, 3849+10 kb C---+T,6,7 which is located in the 6-2 kb EcoRI fragment, 10 kb from the 5’ junction of

Leroy W Matthews Cystic Fibrosis Center, Departments of Pediatrics (R C Stern MD, C F Doershuk MD, M Drumm PhD), and Genetics (M Drumm), Case Western Reserve University; and University Hospitals of Cleveland, Rainbow Babies and Childrens Hospital, Cleveland, Ohio Correspondence to: Dr Robert C Stern, Rainbow Babies and Childrens Hospital, 11 100 Euclid Avenue, Cleveland, OH 44106, USA

274

5

intron 19, and mutation seems

for 1-4% of CF alleles. This to be associated with milder disease.6,1 Understanding of how CF is modified in these patients elucidate the pathophysiology and suggest may interventional strategies. accounts

Methods our patients (406) have been elucidated (Integrated Genetics, Framingham, Massachusetts, USA).8 DNA probes allowed identification of AF508 and 31 other mutations. Ten patients were found to have 3849+10 kb C-T. CF was diagnosed on clinical grounds: raised sweat chloride concentration,9 and either typical pulmonary or digestive signs or a positive family history. For patients with intermediate sweat chloride concentrations (normal <40 mmol/L, intermediate 40-60 mmol/L, abnormal >60 mmol/L), the diagnosis was confirmed by genotyping or by analysis of restriction fragment length polymorphism (RFLP). Respiratory cultures and semen analyses were carried out by hospital laboratories. Other

The genotypes of all

information was obtained by review of records and interviews. Ethics commitee approval and patient or family consent was obtained.

Patients with 3849+10 kb CT of the 406 patients were hemizygous for 3849+10 kb C—T, none was homozygous. The eight children came from six (1-6%) of 372 families. In five families with 3849+10 kb C-T the other CF gene was

Eight (1-9%)

*Age at death. tParent confirmed to have 3849+10 C-T mutation. tNot diagnosed before death from chronic pseudomonas pneumonitis. §No positive Eight isolated positive cultures out of 31 done since diagnosis; does not yet satisfy criteria for chronic colonisation.

cultures

yet.

F=AF508; 3849=3849+10 kb C-T; ND=not done. Table: Characteristics of subjects with 3489+10 kb C---.T

AF508, and in

the other CF mutation remains families an affected sibling had died,

one

unknown. In two and data for these subjects are included (table). No patient required exogenous pancreatic enzymes. For example, trypsin, and chymotrypsin concentrations were 377 and 153 jjbg/g stool in patient 2 and 202 and 254 fJvg/g stool in patient 4 (normal >60 fJvg/g stool for both enzymes). Patients 4 and 10 had pancreatitis. At 5 had microscopic pancreatic necropsy, patient abnormalities but before death he had shown 94% fat absorption by the radioactive triolein method. No patient had meconium ileus, detectable liver disease, or diabetes. Although onset of pulmonary symptoms was late in most of these patients, three developed chronic cough before the age of 4. Two died of pulmonary disease and one needed lung transplantation. Two patients have had no positive pseudonomas cultures yet, and patient 8 has had several positive cultures but does not satisfy our criteria for chronic colonisation (three consecutive positive cultures). The remainder have chronic colonisation. The parents who carried the 3849+10 kb C---+T allele each had at least one ancestor from eastern Europe, Russia, or Italy (ancestry was unknown in one family). No

subject was Jewish. Patient 5 died aged 17 before CF was suspected; his genital tract was normal at necropsy. The other three male patients have had semen analyses. Two brothers (patients 2 and 3) had oligospermia (sperm counts at ages 32 and 27-years, respectively, 53 and 12X 106/mL); sperm morphology was normal. Before he died from pulmonary complications patient 3 fathered a child not affected by CF who is heterozygous for AF508. Patient 2 is unmarried and has no children. The fourth male subject (patient 7) is unmarried. Although he does not meet all the clinical criteria for CF, his sister (patient 6) had pansinusitis and nasal polyps during childhood, and received a double lung transplant because of pseudomonas pulmonary disease. Both siblings have intermediate sweat chloride, concentrations. RFLP analysis confirmed that they inherited the same chromosome 7s (with <1% probability of error). Maternal chromosomes were discriminated by 3’11/Msp, KM19/ Pst 1, SV2C/Taql, and Met H/Msp1 polymorphisms; one

paternal chromosome carried 3849+10 kb C-+T. Thus, both patients have genotype unknown/3849+10 kb C-+T. At age 19, patient 7’s sperm count (198X106/mL) and sperm morphology (by light microscopy) were normal. Discussion Production of some normal protein by genes with a splice mutation can occur-for example in &bgr;-thalassaemia.1O Our data are consistent with Highsmith and colleagues’ hypothesis7 that even if small amounts of normal CF transmembrane conductance regulator (CFTR) are present, the disease is less severe. Mild disease could also result if CFTR encoded by 3849+10 kb C--+T, predicted to be truncated protein, is more functional than that encoded by AF508. CFTR migration to the cell membrane may require or be facilitated by the formation of dimers or other multimers; if the 3849+10 kb C-4T CFTR is a normally functioning and normally transported protein, it could also help to prevent severe disease by transferring abnormal CFTR (eg, AF508) to its functional location. In these and similar patients, increasing overall gene expression or protein activity may produce sufficient function to lead to control of disease, if the abnormal CFTR is inactive or harmless. Semen analysis on our patients and those described by Highsmith et aP suggest that reproductive disease is less severe in patients with 3849+10 kb C—T. This form of CF may lie at the opposite end of a wide spectrum of CF variants from the form described by Anguiano et al," in which obstructive azoospermia seems to be the only manifestation of abnormal genes at the CF locus. Augarten et all’ reported another 3849+10 kb C-T hemizygous patient with a normal spermatogram, and Highsmith et a 17 noted that they had heard about five additional patients who did not have obstructive azoospermia; no data on semen analyses were given, but a one of those patients is father. Although in the male pathophysiological changes reproductive tract are not as severe as in classical CF, our data suggest that they are not invariably eliminated. In individual patients there may ultimately be progression to oligospermia or azoospermia, as suggested by the normal sperm count in our 19-year-old patient, the low counts in our older patients (2 and 3), and the azoospermia in one of 275

Highsmith’s patients.This pathophysiological course could also explain the CF sibship (genotype unknown) in which one brother had azoospermia and the other fathered The

a

child. 13

possibility of altered but not absent pathophysiology would also explain the occasional report of pancreatic insufficiency in AF508/3849+10 kb C-T and the hemizygotes,7 microscopic pancreatic abnormalities found at necropsy in our patient 5. Clinicians should be aware that exocrine pancreatic insufficiency can develop in patients with atypical disease. Although azoopermia is helpful in supporting a CF diagnosis, 14 a normal sperm count or proven fertility is probably less useful in definitively excluding the diagnosis. Longitudinal studies of patients with normal or low sperm will be necessary. Meanwhile, however, sperm banking might be offered to these CF patients, even if counts

they are severely oligospermic. Our patients’ semen analyses and reproductive histories are similar to those described for some patients with Young’s syndrome, in which childhood respiratory disease is also associated with azoospermia. Some Young’s syndrome patients have fathered children before becoming azoospermic.15,16 Perhaps some of these patients will ultimately be found to have other abnormalities at the CF locus. The existence of mild CF phenotype with obstructive azoospermia," and the patients who also have mild phenotype but are not azoospermic suggest some dissociation in the severity of disease between different organs affected by CF. Since the vas deferens is abnormal at birth in classical CF, it is possible that AF508 CFTR brings about early occlusion of the Wolffian duct; CFTR is expressed by the 18th week of gestation, when the vas deferens develops. The vas deferens was thought to be extremely sensitive (and with virtually no heterogeneity) to impaired CFTR activity.2 However, our observations suggest that the abnormality in 3849+10 kb C-T patients is consistently less severe during embryonic development of male genital tract but has the potential to cause obstructive azoospermia later.

276

This work was supported in part by grant DK 27651 from the National Institutes of Health and by grants from the Cystic Fibrosis Foundation and United Way Services of Greater Cleveland.

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