Age of SERPINA1 gene PI Z mutation: Swedish and Latvian population analysis.

Alpha 1-antitrypsin (A1AT) deficiency, one of the most common inborn errors of metabolism in Caucasians, is characterized by a low serum concentration of A1AT and a high risk of pulmonary emphysema and liver disease. The allelic frequency for the most common protease inhibitor (PI) Z mutation in the SERPINA1 gene is 2-5% in Caucasians of European descent. The objective of our study was to estimate the PI Z mutation age using molecular analysis in Latvian and Swedish populations, which have the highest frequency of PI Z mutation. DNA samples of heterozygous and homozygous PI Z allele carriers from Latvia (n = 21) and Sweden (n = 65) were analysed; 113 unrelated healthy donors from Latvia were used as a control group. MALDI-TOF analysis was performed on all samples. Pairwise Fst was computed to compare the PI Z mutation ages between the two populations and controls. A p value less than 0.05 was considered significant. Analysis of non-recombinant SNPs revealed that the PI Z mutation age was 2902 years in Latvia (SD 1983) and 2362 years in Sweden (SD 1614) which correlates with previous studies based on microsatellite analysis.

Plasma neutrophil lipocalin, elastase-alpha1-antitrypsin complex and neutrophil protease 4 in preterm infants with respiratory distress syndrome.

Respiratory distress syndrome (RDS) and chronic lung disease of prematurity (CLD) are associated with inflammation of the airways and interstitial tissue of the lung. It is hypothesized that RDS severity and the risk of developing CLD may be correlated with neutrophil gelatinase-associated lipocalin (NGAL), a marker of leucocyte activity, human elastase-alpha1-antitrypsin complex (HEAT) or free and complexed neutrophil protease 4 (NP4), markers of proteolytic enzyme secretion from granulocytes. Thirty-three preterm infants with RDS were enrolled in the study and plasma sampled between 3 and 14 days of life. NGAL, HEAT and NP4 concentrations varied widely in infants with RDS. Significant correlations between subsequent development of CLD and plasma concentrations of HEAT and NP4, respectively, were found on days 3-4 of life, p=0.006 and p=0.02, respectively.

Tracheobronchial aspirate fluid neutrophil lipocalin, elastase- and neutrophil protease-4-alpha1-antitrypsin complexes, protease inhibitors and free proteolytic activity in respiratory distress syndrome.

UNLABELLED: This study aimed to determine whether the protease/protease inhibitor balance and neutrophil activity is of pathophysiological importance in the severity and resolution of respiratory distress syndrome (RDS) and the eventual development of neonatal chronic lung disease (CLD). Ventilated preterm infants with RDS (n = 43) were studied during their first week of life. Tracheobronchial aspirate fluid (TAF) concentrations of neutrophil lipocalin, the elastase- and neutrophil protease-4 (NP4) complex concentrations, and alpha1-antitrypsin (alpha1AT), antichymotrypsin (ACT) and secretory leucocyte protease inhibitor (SLPI) levels were analysed. Free proteolytic and elastolytic activities were also determined. CLD correlated with low alpha1AT (p = 0.02) and ACT (p = 0.02) levels at 3-4 d of age and low SLPI (p = 0.03) at 7-8 d of age. No correlations were found between CLD or severity of RDS (as judged from radiological examination) and neutrophil lipocalin, elastase- and NP4-alpha1AT complexes during the first week of life, with one exception: RDS X-ray severity and the elastase-alpha1AT complex concentration were correlated at 3-4d of age (p = 0.02). Free proteolytic activity occurred in the TAF of 7/30 infants tested on day 3-4 and free elastolytic activity in 1 patient. During the rest of the first week of life no free elastolytic or proteolytic activities were observed. Caesarean section was correlated with low levels of SLPI on day 3-4 (p = 0.01), NP4 (p = 0.03) and ACT (p = 0.05) on day 5-6. Gestational age was positively correlated with protease inhibitors and their complexes at 3-4 d of age. CONCLUSION: Free proteolytic or elastolytic activity in the TAF of RDS infants in the first week of life occurred by way of exception. Elastase-/NP4-alpha1AT complex or neutrophil lipocalin levels were not correlated with the development of CLD. The correlation between CLD and low alpha1AT or ACT at 3-4 d and SLPI at 7-8 d of age may be due to either immaturity or complex formation. The severity of RDS as judged from radiological examination was correlated with elastase-alpha1AT complex on day 3-4. The main hypothesis, that TAF protease/protease inhibitor levels or imbalance and leucocyte activity are important factors indicating a high risk of severe RDS and subsequent CLD development, was principally not confirmed.

A longitudinal cohort study on the prevalence of Helicobacter pylori antibodies in Swedish children and adolescents.

UNLABELLED: The aim of this study was to monitor the Helicobacter pylori antibody seroprevalence of an asymptomatic cohort between the ages of 4 and 18 y. The H. pylori antibody titres in a longitudinally followed cohort of 168 native Swedish children (born between 1972 and 1974) were established at 4, 8, 12, 16, and 18 y of age. Seventeen children (10.1%) were found positive on at least one occasion when a paediatric cut-off was applied. Five children (3.0%) reached levels considered positive in adults. The seroprevalence at 4 y of age was 4.0%, at 8 y 2.5%, at 12 y 4.9%, at 16 y 5.3%, and at 18 y 6.3%. The difference in serological titres between the age groups was not significant. A change from negative to positive after the age of 4 took place in 5 of the cases. Spontaneous seroreversion appeared in 5 cases. CONCLUSION: Our findings showed no significant differences among the various age groups. Seventeen of the 168 children (10.1%) had been infected at some time, the prevalence ranging from 2.5% to 6.3%. Seroconversion and seroreversion occurred infrequently between the ages of 4 and 18 y.

Respiratory symptoms and lung function in young adults with severe alpha(1)-antitrypsin deficiency (PiZZ).

BACKGROUND: Neonatal screening for alpha(1)-antitrypsin (AAT) deficiency was undertaken in Sweden between 1972 and 1974 when 129 infants with severe AAT deficiency (phenotype PiZ) were identified. The cohort has been followed up prospectively. METHODS: 124 PiZ subjects, still alive and still living in Sweden, were invited to a follow up examination at about 22 years of age. The check up included a clinical examination, spirometric tests, and a questionnaire on smoking habits and respiratory symptoms. RESULTS: Ninety eight subjects (97 PiZZ and 1 PiZ-) subjects attended the follow up. The mean age of the subjects was 22.5 years (range 19.8-24.8). The mean (SD) forced expiratory volume in 1 second (FEV(1)) was 98 (14)% predicted, vital capacity (VC) was 103 (14)% predicted, and the mean FEV(1)/VC ratio was 83 (7)%. Eighty six subjects had previously undergone spirometric tests. The median follow up time was 4.3 years (range 0.9-7.3). The mean annual change in FEV(1) (% predicted) was -1.2% (95% CI -2.1 to -0.3), in VC (% predicted) was -1.5% (95% CI -2.0 to -0.9), and in the FEV(1)/VC ratio (%) was -0.3% (95% CI -0.7 to 0.2). Twenty eight individuals (29%) reported recurrent wheezing. Fifteen subjects (15%) had been diagnosed by a physician as having asthma. Eighteen subjects reported that they had smoked at some time; 10 were current smokers. The mean number of pack years among the ever smokers was 3.4 (range 0.6-10.5). Ten of 18 ever-smokers and 18 of 80 non-smokers reported recurrent wheezing (p<0.01), while exertional dyspnoea was reported by six ever smokers and 11 non-smokers (p<0.05). Lung function test results did not differ significantly between ever smokers and non-smokers. CONCLUSIONS: Young PiZ adults have essentially normal lung function, but have a high prevalence of asthma symptoms. Smoking in these individuals is associated with an increased frequency of respiratory symptoms.

A future for neonatal alpha1-antitrypsin screening?

A WHO expert group recommends neonatal screening for alpha1-antitrypsin deficiency (alpha1ATD). Homozygous alpha1ATD PiZZ occurs in 1 in 5,000 of the U.S. Caucasian population and up to 1 in 500 individuals of the European population, with a large regional variation. It is a risk factor that predisposes mainly to liver disease in early infancy and emphysema in early adulthood. Most importantly, smoking decreases the duration of the asymptomatic phase and life expectancy by 10-20 y. The Swedish alpha1AT screening programme and subsequent information and advice prevented the majority of adolescents from starting to smoke. The involved parents and alpha1ATD adolescents retrospectively recommended neonatal screening. Potential advantages of neonatal alpha1AT screening are: early diagnosis and treatment of neonatal liver disease, optimal treatment of fever and bacterial infections theoretically preventing liver cell damage, genetic advice and information about the consequences of passive and active smoking. Potential advantages of postponing screening until age 11-12 y are: identification of alpha1ATD close to the age when smoking may start, and possibility for the child to take part in the screening decision. Disadvantages of alpha1AT screening are: psychosocial reactions-the mother probably being most vulnerable in the neonatal period-and discrimination by insurance companies and employers. Important uncertainties are: lack of knowledge concerning participation in a voluntary alpha1AT screening, psychosocial reactions and the efficacy of anti-smoking advice if the information is given to school-age children and families. Thus the question whether and when to screen for alpha1ATD is still the topic of lively debate.

Expression of an LDL receptor allele with two different mutations (E256K and I402T).

AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effects of the mutations on LDL receptor function. RESULTS: The two mutations were located on the same allele of the LDL receptor gene. All mutant constructs resulted in the production of a detectable protein in CHO cells. The cells expressing only the I402T mutation, or the combination of I402T and E256K mutations, were seriously affected in mediating uptake and degradation of LDL. Contrary to initial predictions, the cells expressing only the E256K mutation showed essentially the same binding, uptake, and degradation of 125I labelled LDL as cells transfected with normal LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation, and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients studied. CONCLUSIONS: Despite the information available on the structure-function relations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitution of E256K alone is a rare sequence variation, without a detectable phenotype modulating effect.

A future for neonatal alpha1-antitrypsin screening?

A WHO expert group recommends neonatal screening for alpha1-antitrypsin deficiency (alpha1ATD). Homozygous alpha1ATD PiZZ occurs in 1 in 5000 of the U.S. Caucasian population and up to 1 in 500 individuals of the European population, with a large regional variation. It is a risk factor that predisposes mainly to liver disease in early infancy and emphysema in early adulthood. Most importantly, smoking decreases the duration of the asymptomatic phase and life expectancy by 10-20 y. The Swedish alpha1AT screening programme and subsequent information and advice prevented the majority of adolescents from starting to smoke. The involved parents and alpha1ATD adolescents retrospectively recommended neonatal screening. Potential advantages of neonatal alpha1AT screening are: early diagnosis and treatment of neonatal liver disease, optimal treatment of fever and bacterial infections theoretically preventing liver cell damage, genetic advice and information about the consequences of passive and active smoking. Potential advantages of postponing screening until age 11-12y are: identification of alpha1ATD close to the age when smoking may start, and possibility for the child to take part in the screening decision. Disadvantages of alpha1AT screening are: psychosocial reactions--the mother probably being most vulnerable in the neonatal period--and discrimination by insurance companies and employers. Important uncertainties are: lack of knowledge concerning participation in a voluntary alpha1AT screening, psychosocial reactions and the efficacy of anti-smoking advice if the information is given to school-age children and families. Thus the question whether and when to screen for alpha1ATD is still the topic of lively debate.

Hereditary dyslipidemias and combined risk factors in children with a family history of premature coronary artery disease.

AIM: Schoolchildren aged 10-11 with a family history of premature coronary artery disease (CAD), were examined in order to identify children with genetically determined dyslipidemias and a combination of risk factors. METHODS: A total of 4000 questionnaires were distributed by the school; 55% of the families answered and returned the questionnaire. Blood lipids, apolipoprotein B, and Lp(a) lipoprotein were analysed in high risk children and their parents. RESULTS: A family history of premature CAD in parents or grandparents was identified in 208 families; 175 agreed to take part in a clinical examination and laboratory tests. Normal blood lipid tests were found in 89 children. Another 48 had an isolated increase of Lp(a) lipoprotein of minor clinical importance. Of the remaining 38 children, 23 had non-hereditary abnormalities of low (LDL) or high density lipoprotein (HDL) cholesterol or apolipoprotein B. Fifteen children were suspected to have genetically determined dyslipidemias or a combination of risk factors: in four, possible familial hypercholesterolaemia (FH); in five, possible familial combined hyperlipidaemia; in three, hereditary low HDL cholesterol; and in three a combination of high LDL cholesterol and Lp(a) lipoprotein concentrations. In addition, possible FH was detected in eight of the parents. CONCLUSION: It is worthwhile asking parents about the occurrence of premature CAD among their child's closest relatives.

Maternal distress and congenital malformations: do mothers of malformed fetuses have more problems?

As compared with 580 randomly chosen pregnant women without malformed offspring. 161 women with malformed offspring at the index pregnancy had a more frequent history of previous multiple offspring deaths and somewhat increased maternal age but were not different on social class, marital or cohabitation status or parity. As compared with demographically similar reproducing women (n = 54) interviewed, malformation cases (n = 98) reported having had significantly more strong stress before identification of the malformation, as well as a clear tendency toward less appropriate timing of the pregnancy. Women with malformed offspring represent a psychosocially vulnerable group and should receive special clinical and personal support.

Neonatal alpha1-antitrypsin screening: parents' views and reactions 20 years after the identification of the deficiency state.

During 1972-74, 200000 Swedish neonates were screened for alpha1-antitrypsin deficiency (alpha1ATD). The parents of the 22-23-y-old alpha1ATD children were asked about their views on the screening study, information and follow-up of their children as well as physical and psychosomatic problems, which were compared with a matched control (MC) group. Of the original 122 alpha1ATD and MC parents, 85 alpha1ATD and 89 MC parents decided to participate. The neonatal period was considered the best time for screening by 88% of the responding parents. The care of the alpha1ATD individuals was assessed as positive or very positive by 84% of the parents. About 70% of the parents thought that the awareness of their child's alpha1ATD had affected their lives, the most common comment being an increased consciousness of smoky and dusty environments. No significant difference between alpha1ATD and control parents was found concerning worry about the child's health and future. When asked about symptoms related to their mental health, no difference was found concerning headache, sleep problems, stomach troubles or tiredness. Alpha1ATD mothers had statistically significantly more anxiety than control mothers (chi2 p<0.01, Wilcoxon p<0.02). The knowledge derived from the present and previous parts of the alpha1AT screening study is important with regard to presymptomatic testing for adult-onset disease in children.

Effect of environmental and clinical factors on lung function and respiratory symptoms in adolescents with alpha1-antitrypsin deficiency.

Individuals identified in the Swedish neonatal alpha1-antitrypsin (AAT) screening study were followed prospectively from their first to their eighteenth year of life. The aim of this study was to analyse the effect of environmental factors, i.e. active and passive smoking, and of clinical factors on lung function and the occurrence of respiratory symptoms in AAT-deficient adolescents. The study group consisted of 88 protease inhibitor (Pi)ZZ and 40 PiSZ adolescents. Medical history including respiratory symptoms, and active and passive smoking were recorded at each follow-up up to the age of 18 y. Lung function tests were performed at the present check-up. At the age of 18 y, both forced expiratory volume in one second (FEV ) and FEV1/vital capacity (VC) were significantly lower in the smoking than in the non-smoking subgroup, and significantly more smokers than non-smokers reported the presence of phlegm. The mean FEV1/VC ratio was lower for those presently exposed to parental smoking. Multiple linear regression analysis indicated that clinical liver disease in early life, active smoking and parental smoking were independent determinants of FEV1/VC. The results suggest that marginal deviations in lung function and the symptom of phlegm among AAT-deficient adolescents occur characteristically early in the subgroup of smokers. Parental smoking may contribute to decreased lung function.

Adolescents with alpha1-antitrypsin deficiency have high alpha2-macroglobulin and low neutrophil lipocalin and elastase levels in plasma.

Eighteen-year-old adolescents with alpha1-antitrypsin (alpha1AT) deficiency have mostly normal lung function tests. We hypothesized that compensatory increases in other protease inhibitors and/or a decreased leukocyte activity might favorably affect the protease/protease-inhibitor balance in alpha1AT-deficient adolescents. At the age of 18 y 46 PiZZ (severe deficiency), 22 PiSZ (moderate deficiency), and 41 control subjects were studied. The plasma protease inhibitors alpha2-macroglobulin (alpha2M), alpha1-antichymotrypsin (Achy), and secretory leukocyte protease inhibitor (SLPI) were studied, and the protease elastase complexed with alpha1AT (HEAT) and neutrophil gelatinase-associated lipocalin (NGAL) as indicators of neutrophil leukocyte activity. Significantly higher concentrations of alpha2M were found in PiZ (p < 0.0001) and PiSZ (p < 0.0001) individuals compared with control subjects. The PiZZ and SZ adolescents had low levels of NGAL (p < 0.0001). Low levels of HEAT were found in PiZZ subjects (p < 0.0005). Higher concentrations of Achy were found in PiZZ (p < 0.04) and PiSZ (p < 0.05) individuals. Increased concentrations of alpha2M and Achy combined with decreased levels of HEAT and NGAL, indicating decreased leukocyte activity may, to some extent, compensate for the protease/protease inhibitor imbalance in the alpha1AT-deficiency state.

Young adults with alpha 1-antitrypsin deficiency identified neonatally: their health, knowledge about and adaptation to the high-risk condition.

The psychological and psychosocial consequences of screening for alpha 1-antitrypsin deficiency (alpha 1 ATD) were investigated when the subjects were 5-7 years old. The present study was conducted when the subjects were 18-20 years old, the foci of interest being their health, psychosomatic problems, knowledge about alpha 1 ATD and the potential effect of that knowledge on their lives and future family planning. Samples of 61 PiZ and 61 demographically matched control subjects, 18-20 years old, were asked to participate. Written, structured questionnaires covered the following items: basic familial characteristics, psychosomatic symptoms, opinions on medical check-ups, information and views on future alpha 1 ATD screening, whether the knowledge about alpha 1 ATD had affected the life and family planning of alpha 1 ATD individuals. Items concerning the "alpha 1 ATD matter" were excluded in the questionnaires given to the controls. Questionnaire data were obtained from 50 alpha 1 ATD and 48 control individuals, 41 of each being matched alpha 1 ATD-control pairs. No significant differences were found in demographic or educational backgrounds, psychosomatic complaints such as headache, sleep difficulties, stomach ache, tiredness or anxiety. Lung symptoms occurred more frequently in alpha 1 ATD subjects (p = 0.05). Six per cent of the alpha 1 ATD individuals planned working careers with a high risk of air pollution. The majority (86%) of the alpha 1 ATD subjects perceived the contact with the medical services as positive; 14% as both positive and negative. The information concerning alpha 1 ATD was assessed as satisfactory by 73%, as both good and bad by 17% and as unsatisfactory by 10%. All alpha 1 ATD subjects advocated general screening for alpha 1 ATD, the neonatal period being chosen as optimal by 94%. Half of the alpha 1 ATD individuals thought that the knowledge of their high-risk condition had affected their lives, particularly their awareness of the dangers of smoking and environmental pollution. The majority, 88%, knew that they should avoid smoking to protect their lungs. In conclusion, no negative psychosocial consequences of the neonatal alpha 1 AT-screening were found in early adulthood. The alpha 1 ATD individuals were aware of the dangers of smoking and were of the opinion that alpha 1 AT-screening should be recommended.

Primary prevention in a high-risk group: smoking habits in adolescents with homozygous alpha-1-antitrypsin deficiency (ATD).

The serious form of alpha-1-antitrypsin deficiency (ATD) Pi ZZ strongly predisposes the individual for pulmonary emphysema and premature death in adulthood, especially if exposed to tobacco smoking. General screening of all new-born children was conducted in Sweden during 1972-1974, the major purpose being to reduce exposure of the child to parental smoking while growing up and to prevent the child from starting to smoke. Sixty-one children with ATD neonatally identified through mass-screening, and their families, have been compared with a demographically matched control group regarding smoking habits, as studied through interviews and questionnaires on two occasions. When the children were 5-7 years old, the smoking rates among parents of the ATD children and especially among the ATD fathers exceeded smoking rates for controls. Thirteen years later no differences in parental smoking were found between the groups. At 18-20 years of age the ATD children reported smoking significantly less than the control children (p < 0.05). From the perspective of prevention, the goal of the neonatal screening to reduce the smoking rates among the parents of the ATD children was not attained, while it was achieved among the ATD children. The results indicate that a screening program with early detection of ATD effectively prevents adolescent children from starting to smoke. From ethical, medical and psychological points of view, a voluntary screening program for ATD in pre-adolescence is recommended.

Reovirus type 1 associated with meningitis.

A previously healthy 3-month-old girl presented with symptoms of meningitis, diarrhoea, vomiting and fever. Green monkey kidney (GMK) cells inoculated with cerebrospinal fluid (CSF) revealed reovirus-like particles by electron microscopy. RNA-gel electrophoresis, immunofluorescence and virus neutralization identified the pathogen isolated from CSF as reovirus type 1. Antibody determination by immunofluorescence of paired sera showed a significant antibody titre rise to the CSF isolate and neutralization test revealed a greater than 4-fold antibody titre rise to the CSF isolate. The CSF isolate was also neutralized by reovirus type 1 antibodies. This report represents one of a few associating reovirus with CNS symptoms in humans.

The liver in adolescents with alpha 1-antitrypsin deficiency.

Of 200,000 Swedish infants screened for alpha 1-antitrypsin deficiency (alpha 1 ATD), 184 (127 PiZ, 2 PiZ-, 54 PiSZ, and 1 PiS-) children have been followed prospectively, of whom 1 PiSZ and 5 PiZ children died in early childhood. We now report clinical and biochemical signs of liver disease in adolescence and the prognosis of neonatal liver disease up to the age of 18 years. The alpha 1 ATD subjects were offered a clinical checkup and liver tests at 16 and 18 years of age, 150 of 178 alpha 1ATD subjects undergoing checkups at age 16 and 166 at age 18. Liver tests were performed in 121 adolescents at both the 16- and 18-year checkups. None of the PiZ and PiSZ subjects checked at the age of 16 and 18 years had any clinical signs of liver disease. Abnormalities of serum alanine aminotransferase (S-ALAT) or gamma-glutamyl transferase (S-GT) were found at the 16-year checkup (all PiZ and PiSZ subjects tested included) in 17% of PiZ and 8% of PiSZ adolescents, and at the age of 18 years in 12% of PiZ and 15% of PiSZ subjects. In only two cases were both S-ALAT and S-GT concentrations abnormal at both the 16-year and 18-year follow-ups. Serum procollagen III peptide concentrations were normal in all those with abnormal liver test results. Of 127 PiZ subjects, 22 had manifested clinical signs of liver disease in infancy. Of these 22, two died early in life of cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)

An efficient screening procedure detecting six novel mutations in the LDL receptor gene in Swedish children with hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal semi-dominant disorder caused by defects in the low density lipoprotein receptor (LDLR) gene and is a well-documented risk factor for developing cardiovascular disease. The LDLR genes of five Swedish children with FH were examined in this study. Initial mutation screening was performed by denaturing gradient gel electrophoresis (DGGE) with enzymatically amplified exon-sized fragments, each containing a tailing GC-rich requence. The GC-clamped fragments had been synthesized with a restriction site adjacent to the intron-corresponding sequence to allow detachment of the clamps, thereby rendering the fragments suitable for subsequent analysis by single-strand conformation polymorphism (SSCP) analysis of samples from patients with no DGGE-detectable mutations. In addition, all the LDLR genes of the patients were screened for large alterations by restriction fragment length polymorphism analysis. Following this strategy, seven different, potentially disease-causing mutations were detected in the five children with FH. Six of the alterations, five single-base substitutions and one dinucleotide deletion, have not previously been described. DGGE detected six of the mutations and SSCP the seventh.