RESUMEN
Patients with classical galactosaemia (galactose-1-phosphate uridyltransferase (GALT) deficiency) manifest clinical complications despite strict dietary galactose restriction. Therefore the significance of endogenous galactose production has been assessed. Previous in vivo studies primarily focused on patients homozygous for the most common genetic variant Q188R but little is known about other genetic variants. In the present study the endogenous galactose release in a group of non-Q188R homozygous galactosaemic patients (n = 17; 4-34 years) exhibiting comparably low residual GALT activity in red blood cells was investigated. Primed continuous infusion studies with D-[1-(13)C]galactose as substrate were conducted under post-absorptive conditions and in good metabolic control. The results demonstrate that all patients exhibiting residual GALT activity of <1.5% of control showed a comparable pathological pattern of increased endogenous galactose release irrespective of the underlying genetic variations. Possible implications of the findings towards a more differentiated dietary regimen in galactosaemia are discussed.
Asunto(s)
Galactosa/biosíntesis , Galactosemias/metabolismo , UTP-Hexosa-1-Fosfato Uridililtransferasa/deficiencia , Adolescente , Adulto , Niño , Preescolar , Eritrocitos/enzimología , Femenino , Galactosa/metabolismo , Galactosemias/sangre , Galactosemias/enzimología , Humanos , Masculino , Adulto JovenRESUMEN
BACKGROUND: Indirect calorimetry (IC) with metabolic monitors is widely used for noninvasive assessment of energy expenditure and macronutrient oxidation in health and disease. OBJECTIVE: To overcome deficiencies in validity and reliability of metabolic monitors, we established a procedure that allowed correction for monitor-specific deviations. DESIGN: Randomized comparative IC (canopy mode) with the Deltatrac MBM-100 (Datex) and Vmax Encore 29n (SensorMedix) was performed in postabsorptive (overnight fast >8 h) healthy subjects (n = 40). In vitro validation was performed by simulation of oxygen consumption (VO2) and carbon dioxide output (VCO2) rates by using mass-flow regulators and pure gases. A simulation-based postcalorimetric calibration of cart readouts [individual calibration control evaluation (ICcE)] was established in adults (n = 24). RESULTS: The comparison of carefully calibrated monitors showed marked differences in VCO2 and VO2 (P < 0.01) and derived metabolic variables [resting energy expenditure (REE), respiratory quotient (RQ), glucose/carbohydrate oxidation (Gox), and fat oxidation (Fox); P < 0.001]. Correlations appeared to be acceptable for breath gas rates and REE (R(2) ~ 0.9) but were unacceptable for RQ (R(2) = 0.3), Gox, and Fox (R(2) = 0.2). In vitro simulation experiments showed monitor-dependent interferences for VCO2 and VO2 as follows: 1) within series, nonlinear and variable deviations of monitor readouts at different exchange rates; 2) between series, differences and unsteady variability; and 3) differences in individual monitor characteristics (eg, rate dependence, stability, imprecision). The introduction of the postcalorimetric recalibration by ICcE resulted in an adjustment of gas exchange rates and the derived metabolic variables with reasonable correlations (R(2) > 0.9). CONCLUSIONS: Differential, metabolic, monitor-specific deviations are the primary determinants for lack of accuracy, comparability, and transferability of results. This problem can be overcome by the present postcalorimetric ICcE procedure.
Asunto(s)
Calibración , Calorimetría Indirecta/métodos , Metabolismo , Monitoreo Fisiológico/métodos , Adulto , Metabolismo Basal , Calorimetría Indirecta/instrumentación , Metabolismo de los Hidratos de Carbono , Dióxido de Carbono/metabolismo , Ayuno , Humanos , Metabolismo de los Lípidos , Persona de Mediana Edad , Monitoreo Fisiológico/instrumentación , Consumo de Oxígeno , Reproducibilidad de los Resultados , RespiraciónRESUMEN
Several mechanisms, such as innate immune responses via Toll-like receptor-4, accumulation of diacylglycerols (DAG)/ceramides, and activation of protein kinase C (PKC), are considered to underlie skeletal muscle insulin resistance. In this study, we examined initial events occurring during the onset of insulin resistance upon oral high-fat loading compared with lipid and low-dose endotoxin infusion. Sixteen lean insulin-sensitive volunteers received intravenous fat (iv fat), oral fat (po fat), intravenous endotoxin (lipopolysaccharide [LPS]), and intravenous glycerol as control. After 6 h, whole-body insulin sensitivity was reduced by iv fat, po fat, and LPS to 60, 67, and 48%, respectively (all P < 0.01), which was due to decreased nonoxidative glucose utilization, while hepatic insulin sensitivity was unaffected. Muscle PKCθ activation increased by 50% after iv and po fat, membrane Di-C18:2 DAG species doubled after iv fat and correlated with PKCθ activation after po fat, whereas ceramides were unchanged. Only after LPS, circulating inflammatory markers (tumor necrosis factor-α, interleukin-6, and interleukin-1 receptor antagonist), their mRNA expression in subcutaneous adipose tissue, and circulating cortisol were elevated. Po fat ingestion rapidly induces insulin resistance by reducing nonoxidative glucose disposal, which associates with PKCθ activation and a rise in distinct myocellular membrane DAG, while endotoxin-induced insulin resistance is exclusively associated with stimulation of inflammatory pathways.
Asunto(s)
Resistencia a la Insulina/fisiología , Lípidos/farmacología , Hígado/metabolismo , Músculo Esquelético/metabolismo , Administración Intravenosa , Administración Oral , Adulto , Glucemia/metabolismo , Calorimetría Indirecta , Femenino , Humanos , Insulina/metabolismo , Interleucina-6/metabolismo , Lípidos/administración & dosificación , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Masculino , Músculo Esquelético/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6.
Asunto(s)
Exosomas/química , Galactosemias/orina , Glicoproteínas de Membrana/orina , Polisacáridos/química , Adulto , Estudios de Casos y Controles , Femenino , Galactosemias/metabolismo , Glicómica , Glicosilación , Humanos , Masculino , Manosa , Glicoproteínas de Membrana/química , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Uromodulina/orinaRESUMEN
Galactokinase deficiency (GALK-D), an autosomal recessive disorder in the Leloir pathway, results in accumulation of galactose, galactitol, and galactonate and leads to early onset of juvenile bilateral cataract. Highest incidence of GALK-D is found in Romani populations. The migration wave due to the Yugoslavian civil war has changed the spectrum of inborn errors of metabolism within Europe. Hence, newborn screening (NBS) in the Berlin region, performed from 1991 until 2010 in 683,675 neonates, revealed an increased incidence of GALK-D of 1:40,000, comparable to that of galactose-1-phosphate-uridyltransferase deficiency. A total of 44% of GALK-D patients were of Romani origin. All patients of Bosnian or Serbian origin were homozygous for the Romani founder mutation p.P28T. Detection of GALK-D by NBS and early start of galactose-restricted diet resulted in regression or prevention of cataracts. Slight cataracts without visual impairment occurred in 50% of the patients, 56% of whom were noncompliant. Further clinical symptoms, e.g., hypoglycemia, mental retardation, microcephaly, and failure to thrive, were associated with noncompliance. With treatment, galactose in blood decreased from 8,892 ± 5,243 to 36.5 ± 49.3 µmol/l, galactose in urine from 31,820 ± 32,103 to 30.0 ± 36.1 µmol/mmol creatinine, galactitol in RBC from 1,584 ± 584 to 12.3 ± 9.4 µmol/l, and galactitol in urine from 11,724 ± 4,496 to 236 ± 116 µmol/mmol creatinine. This is the first presentation of outcome and clinical features in GALK-D patients diagnosed by NBS. As our data suggest, GALK-D should be considered for inclusion in NBS in populations expected to have substantial numbers of GALK-D carriers, e.g., Yugoslavian immigrants.
Asunto(s)
Galactosemias , Bosnia y Herzegovina , Emigración e Inmigración , Femenino , Galactitol/metabolismo , Galactosa/metabolismo , Galactosemias/diagnóstico , Galactosemias/etnología , Galactosemias/terapia , Alemania , Homocigoto , Humanos , Incidencia , Recién Nacido , Masculino , Mutación , Tamizaje Neonatal/métodos , Serbia , Resultado del TratamientoRESUMEN
OBJECTIVE: To conduct a longitudinal assessment of long-term cognitive outcome in patients with classical galactosemia. METHODS: Inclusion criteria were (1) previous assessment of IQ dating back >10 years with tests being comparable with the recent German tests HAWIK-III and HAWIE-R, (2) absence of illnesses other than galactosemia, (3) absence of foreign language problems, (4) enzymatic-metabolic proof of classical galactosemia, (5) compliance with dietary therapy, and (6) written informed consent. Twenty-three patients who fulfilled these criteria were found. They underwent the first IQ test at a mean age of 11 +/- 5 years and the second 13.6 to 15.5 years later at a mean age of 26 +/- 5 years. Results were corrected for the obsolescence of test norms (Flynn effect). RESULTS: Mean total IQ scores on the first and second tests were 78 +/- 14 and 73 +/- 15, respectively, and not significantly different. IQ scores in the average range were observed for 7 patients on the first test and for 5 patients on the second test. For 17 patients, the intraindividual IQ scores remained essentially unchanged. Five patients showed a decrease and 1 an increase of the IQ score over time. No consistent pattern of change was found with respect to performance or verbal IQ subscores or in achievements in the individual subtest. CONCLUSIONS: The results confirm the presence of reduced cognitive ability in classical galactosemia and present evidence for an absence of substantial galactosemia-induced aggravation of this impairment with increasing age, at least in patients from 4 to 40 years of age. It remains to be clarified whether a reduction of cognitive function in galactosemia may be initiated by an in utero toxicity of endogenously formed galactose and which role such a process may play in the development of intellectual deficiencies that are later maintained throughout life.
Asunto(s)
Trastornos del Conocimiento/etiología , Galactosemias/complicaciones , Inteligencia , Adolescente , Adulto , Niño , Preescolar , Factores de Confusión Epidemiológicos , Femenino , Estudios de Seguimiento , Humanos , Pruebas de Inteligencia , Masculino , Adulto JovenRESUMEN
Pregnancy, delivery, and postpartal metabolic control was monitored biochemically in five patients (22-38 years of age) with clinically, enzymatically, and genotypically established classical galactosaemia and good dietary compliance. Three of the patients performed breast feeding of their newborns. Monitoring parameters were galactose-1-phosphate and galactitol concentrations in erythrocytes and urinary excretion of galactose, galactitol, galactonate, and lactose. During pregnancy, a small but steady increase of renal metabolite excretion rates was observed. After delivery, a moderate transient increase of metabolite concentrations with peak values within the first week post partum occurred, irrespective of breast feeding. Altogether, there was no evidence for clinically or subclinically significant changes of metabolic control during pregnancy, delivery, or lactation. In conclusion, a specific metabolic monitoring is apparently not required in pregnant galactosemic women, and breast feeding of the nongalactosemic offspring can be recommended.
Asunto(s)
Lactancia Materna , Galactosemias/metabolismo , Lactancia/metabolismo , Complicaciones del Embarazo/metabolismo , Adulto , Cesárea , Eritrocitos/química , Femenino , Galactitol/metabolismo , Galactosa/metabolismo , Galactosafosfatos/metabolismo , Humanos , Embarazo , Resultado del Embarazo , Adulto JovenRESUMEN
BACKGROUND: Two new inborn errors in the pentose phosphate pathway have been described: ribose-5-isomerase deficiency and transaldolase deficiency. These defects are characterized by accumulation of specific polyols in body fluids. Little is known about human polyol metabolism, but there are indications for a physiological role primarily during early development. OBJECTIVES: The objective of this study was to evaluate the urinary excretion of polyols in neonates with special interest on a possible impact of the grade of maturity. For comparison, urinary polyol excretion in older children was also studied. METHODS: Urine samples of 40 neonates born between gestational week 25 and 41 were analyzed for the excretion of pentose phosphate pathway-associated polyols (erythritol, D-arabitol, ribitol, xylitol). These metabolites were also quantified in urine obtained from 77 children aged 4 weeks to 10 years. RESULTS: The results show high urinary polyol excretions after birth independent of the week of gestation. During the first months of life, the concentrations decreased exponentially and reached a fairly stable steady state thereafter. CONCLUSIONS: Urinary excretion of polyols shows an age dependency with highest concentrations postnatally independent of the grade of maturity. These findings suggest a possible connection between the formation of pentose phosphate pathway-associated polyols and fetal development.
Asunto(s)
Vía de Pentosa Fosfato/fisiología , Alcoholes del Azúcar/orina , Factores de Edad , Niño , Preescolar , Femenino , Edad Gestacional , Humanos , Lactante , Recién Nacido , Masculino , UrinálisisRESUMEN
BACKGROUND: Newborn infants are exposed to various sources of oxidative and/or nitrative stress, which refers to either oxidation and/or nitration of endogenous proteins including loss of their original function. Nitrative stress is predominantly caused following synthesis of peroxynitrite. Particularly preterm infants with immature defense mechanisms against free radical injury appear at risk. OBJECTIVE: To test the feasibility of quantifying the degradation products of the peroxynitrite marker nitrotyrosine [3-nitro-4-hydroxyphenylacetic acid (NHPA) and para-hydroxyphenylacetic acid (PHPA)] in neonatal urine samples. METHODS: NHPA and PHPA were determined by gas chromatography/mass spectroscopy in urinary samples of preterm and term infants (mean gestational age = 28.4 and 39.6 weeks, respectively). RESULTS: The urinary NHPA levels were lower in preterm infants in comparison with term infants. When the NHPA levels were adjusted to the urinary PHPA levels, no differences were found between the two groups. CONCLUSIONS: Nitrotyrosine can be quantified in urinary samples of even the most immature infants. Nitration of endogenous PHPA in the gastrointestinal tract of term infants may have masked potentially higher levels of NHPA in preterm infants.
Asunto(s)
Recién Nacido/orina , Recien Nacido Prematuro/orina , Nitrofenoles/orina , Tirosina/análogos & derivados , Radicales Libres/metabolismo , Tracto Gastrointestinal/metabolismo , Edad Gestacional , Humanos , Estrés Oxidativo/fisiología , Oxígeno/metabolismo , Fenilacetatos/orina , Tirosina/metabolismoRESUMEN
Maple syrup urine disease (MSUD) is an inherited deficiency of branched chain alpha-ketoacid dehydrogenase (BCKDH) activity impairing the degradation of the branched chain amino acids valine, leucine, and isoleucine. Classic MSUD may lead to severe neonatal encephalopathy including coma and impaired cognitive outcome in later life. Early start of dietary treatment and careful metabolic control may improve the outcome of patients with classic MSUD. The aim of this study was to investigate the impact of long-term metabolic control assessed by plasma leucine levels on cognitive outcome in patients with classic MSUD. Plasma leucine levels of 24 patients were obtained retrospectively for the first 6 y of life and yearly medians of mean plasma leucine levels were calculated. At the age of 6 y, IQ tests were performed. Yearly medians of mean plasma leucine levels yielded three homogeneous clusters (low, intermediate, high). Patients of the low cluster showed statistically significant higher IQ scores compared with those of those of intermediate and high clusters. Long-term plasma leucine levels are associated with impaired cognitive outcome in patients with classic MSUD. To achieve the best possible intellectual outcome for affected individuals, we recommend that in infants and preschool children the target range for plasma leucine should not exceed 200 micromol/L.
Asunto(s)
Trastornos del Conocimiento/prevención & control , Leucina/sangre , Enfermedad de la Orina de Jarabe de Arce/dietoterapia , Niño , Trastornos del Conocimiento/diagnóstico , Femenino , Humanos , Pruebas de Inteligencia , Masculino , Enfermedad de la Orina de Jarabe de Arce/sangre , Enfermedad de la Orina de Jarabe de Arce/psicologíaRESUMEN
Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder that causes acute and chronic brain dysfunction because of a neurotoxic effect of the accumulating branched chain amino acids (BCAA) and their corresponding keto acids. Aim of the treatment is a rapid reversal of the neonatal decompensation and a stable long-term metabolic control obtained by a carefully adjusted BCAA-low diet. In optimally treated patients, an unimpaired neurological and intellectual outcome is possible. Ten patients of Turkish origin suffering from MSUD are presently treated in the Metabolic Unit of the University Hospital in Düsseldorf, Germany. All patients show mild intellectual deficits; neurological impairment is rare. This paper aims to define the feasible standard of therapy and the resulting intellectual and psychosocial outcome achievable in MSUD patients of Turkish origin under high standard conditions of medical care for inborn errors of metabolism.
Asunto(s)
Leucina/sangre , Enfermedad de la Orina de Jarabe de Arce/epidemiología , Adolescente , Adulto , Niño , Preescolar , Consanguinidad , Femenino , Alemania/epidemiología , Humanos , Inteligencia , Pruebas de Inteligencia , Masculino , Enfermedad de la Orina de Jarabe de Arce/psicología , Enfermedad de la Orina de Jarabe de Arce/terapia , Resultado del Tratamiento , Turquía/etnologíaRESUMEN
A stable isotope dilution assay was developed for the sensitive determination of D-galactonic acid. D-[U-13C(6)]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U-13C-labelled D-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography-mass spectrometry (GC-MS). Positive chemical ionisation and monitoring of the [MH-60](+)-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of D-galactonate excretion in healthy subjects and galactosemic patients and to monitor the D-galactonate-D-galactitol ratio in human urine.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Azúcares Ácidos/orina , Adolescente , Adulto , Isótopos de Carbono , Niño , Preescolar , Femenino , Galactitol/orina , Galactosa/orina , Galactosemias/orina , Humanos , Técnicas de Dilución del Indicador , Lactonas/orina , Masculino , Control de Calidad , Sensibilidad y EspecificidadRESUMEN
The age dependence of endogenous galactose formation was investigated in Q188R homozygous galactosemic patients (n=18; 4-38 years) using the primed continuous infusion approach with D-[1-13C]galactose as a substrate. Studies were conducted under postabsorptive conditions (fasting >10h) and good metabolic control. In the patients, the release of galactose from endogenous sources into plasma (R(a)) decreased with age and ranged from 4.6 to 2.0 micromol/kg body weight per h. Galactitol and galactonate release rates paralleled the galactose R(a) but at a lower level. The mean relation of galactose, galactitol, and galactonate release was 10:5:1. Statistically, there was a highly significant (p<0.0001) inverse correlation between total galactose release (i.e., sum of R(a) plus galactitol and galactonate release) and age. The data (total galactose=y, age=t) were best fitted to the simple exponential model y=y(0)+axexp(-bt) by non-linear regression analysis. The parameter estimates were y(0)=3.0+/-0.2, a=6.5+/-0.4, and b=0.11+/-0.02. The value of y(0) provides an estimate of total galactose release in adult patients (i.e., approximately 13 mg/kg body weight per day), summation operator (y(0)+a) provides an estimate for galactosemic newborns (i.e., approximately 41 mg/kg body weight per day). The data show that significant amounts of endogenous galactose are formed in galactosemic patients with release rates being several fold higher in infants than in adults. The present findings can explain the persistently elevated galactose-1-phosphate levels in erythrocytes-and its age dependence-in galactosemic patients even when under strict dietary treatment.
Asunto(s)
Galactosa/metabolismo , Galactosemias/genética , Mutación , Adolescente , Adulto , Factores de Edad , Estatura , Peso Corporal , Isótopos de Carbono , Niño , Preescolar , Galactitol/sangre , Galactitol/metabolismo , Galactitol/orina , Galactosa/sangre , Galactosa/orina , Galactosemias/metabolismo , Humanos , Bombas de Infusión , Modelos Biológicos , Azúcares Ácidos/sangre , Azúcares Ácidos/metabolismo , Azúcares Ácidos/orina , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
An established gas chromatography/mass spectrometry (GC/MS) method, devised for stable-isotope dilution analysis of plasma galactose, was developed to allow determination of erythrocyte (red blood cell, RBC) concentrations of galactose-1-phosphate and other primary metabolites relevant in galactosaemia. Galactose-1-phosphate was enzymatically converted to galactose, and the aldononitrile pentaacetate derivative was separated by gas chromatography and determined by mass spectrometry using chemical ionisation and selected ion monitoring of the [MH-60](+) ion. U-(13)C-Labelled standard was used for quantification. Comparative measurements were conducted using established fluorimetric and radiometric enzymatic methods. The GC/MS analysis for galactose-1-phosphate was linear (range examined 0-600 micromol/L(RBC), packed cells), of acceptable repeatability at low and high concentrations (within and between run CVs <15%), with a limit of quantification of 0.01 micromol/L(RBC). With samples from patients with classical galactosaemia there was a linear correlation with conventional enzymatic assays (r(2) > 0.927). In erythrocytes from post-absorptive patients under treatment, Q188R-heterozygous parents, and healthy subjects, galactose-1-phosphate concentrations (mean +/- SD) were found to be 142 +/- 38 (n = 41), 1.4 +/- 0.2 (n = 8), and 1.9 +/- 0.5 (n = 33) micromol/L(RBC), respectively. In comparison, free galactose concentrations were 3.8 +/- 1.7, 0.49 +/- 0.19, and 0.43 +/- 0.20 mol/L(RBC), respectively. The procedure allowed simultaneous galactitol analysis and proved to be useful to trace incorporation of (13)C-label into erythrocyte galactose metabolites in a D-[1-(13)C]galactose in vivo turnover study.
Asunto(s)
Análisis Químico de la Sangre/métodos , Eritrocitos/metabolismo , Galactitol/sangre , Galactosa/sangre , Galactosemias/sangre , Galactosafosfatos/sangre , Cromatografía de Gases y Espectrometría de Masas/métodos , Técnica de Dilución de Radioisótopos , Femenino , Galactitol/metabolismo , Galactosa/metabolismo , Galactosemias/diagnóstico , Galactosafosfatos/metabolismo , Humanos , Marcaje Isotópico/métodos , Masculino , Tasa de Depuración Metabólica , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder of panethnic distribution caused by a deficiency of the activity of branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in the human BCKD genes E1alpha (BCKDHA), E1beta (BCKDHB) and E2 (DBT) are known to result in MSUD, referred to as type Ia, Ib and II mutations respectively. In this study 16 patients with the classic severe form of MSUD and three patients with milder variant forms of the disease were investigated for mutations in the E1alpha-, E1beta- and E2-gene by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The patients' clinical and biochemical phenotypes were well characterized. One novel type Ia missense mutation, eight novel type Ib (three missense, two nonsense, two small deletions, one small duplication) and three novel type II (two missense, one splice site) mutations were identified in patients. Moreover, eleven previously described mutations were detected: five type Ia (four missense, one nonsense), three type Ib mutations (two missense, one nonsense) and three type II mutations (two missense, one small deletion). Fourteen patients are homozygous for one single mutation, five patients are compound-heterozygous for two different mutations affecting one of the three genes. Thus, in all 19 patients the identified mutations can most probably be considered the molecular basis of the disease.