Dissection of biochemical borderline phenotypes in carriers and genetic variants of medium-chain acyl-CoA dehyrogenase deficiency: implications for newborn screening [corrected].

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) represents a potentially fatal fatty acid beta-oxidation disorder. Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has been implemented worldwide, but is associated with unresolved questions regarding population heterogeneity, burden on healthy carriers, cut-off policies, false-positive and negative rates. In a retrospective case-control study, 333 NBS samples showing borderline acylcarnitine patterns but not reaching recall criteria were genotyped for the two most common mutations (c.985A>G/c.199C>T) and compared with genotypes and acylcarnitines of 333 controls, 68 false-positives, and 34 patients. c.985A>G was more frequently identified in the study group and false-positives compared to controls (1:4.3/1:2.3 vs. 1:42), whereas c.199C>T was found more frequently only within the false-positives (1:23). Biochemical criteria were devised to differentiate homozygous (c.985A>G), compound heterozygous (c.985A>G/c.199C>T), and heterozygous individuals. Four false-negatives were identified because our initial algorithm required an elevation of octanoylcarnitine (C(8)) and three secondary markers in the initial and follow-up sample. The new approach allowed a reduction of false-positives (by defining high cut-offs: 1.4 micromol/l for C(8); 7 for C(8)/C(12)) and false-negatives (by sequencing the ACADM gene of few suspicious samples). Our validation strategy is able to differentiate healthy carriers from patients doubling the positive predictive value (42-->88%) and to target NBS to MCADD-subsets with potentially higher risk of adverse outcome. It remains controversial, if NBS programs should aim at identifying all subsets of all diseases included. Because the natural course of milder variants cannot be assessed by observational studies, our strategy could serve as a general model for evaluation of MS/MS-based NBS.

Treatment recommendations in long-chain fatty acid oxidation defects: consensus from a workshop.

Published data on treatment of fatty acid oxidation defects are scarce. Treatment recommendations have been developed on the basis of observations in 75 patients with long-chain fatty acid oxidation defects from 18 metabolic centres in Central Europe. Recommendations are based on expert practice and are suggested to be the basis for further multicentre prospective studies and the development of approved treatment guidelines. Considering that disease complications and prognosis differ between different disorders of long-chain fatty acid oxidation and also depend on the severity of the underlying enzyme deficiency, treatment recommendations have to be disease-specific and depend on individual disease severity. Disorders of the mitochondrial trifunctional protein are associated with the most severe clinical picture and require a strict fat-reduced and fat-modified (medium-chain triglyceride-supplemented) diet. Many patients still suffer acute life-threatening events or long-term neuropathic symptoms despite adequate treatment, and newborn screening has not significantly changed the prognosis for these severe phenotypes. Very long-chain acyl-CoA dehydrogenase deficiency recognized in neonatal screening, in contrast, frequently has a less severe disease course and dietary restrictions in many patients may be loosened. On the basis of the collected data, recommendations are given with regard to the fat and carbohydrate content of the diet, the maximal length of fasting periods and the use of l-carnitine in long-chain fatty acid oxidation defects.

Management and outcome in 75 individuals with long-chain fatty acid oxidation defects: results from a workshop.

At present, long-chain fatty acid oxidation (FAO) defects are diagnosed in a number of countries by newborn screening using tandem mass spectrometry. In the majority of cases, affected newborns are asymptomatic at time of diagnosis and acute clinical presentations can be avoided by early preventive measures. Because evidence-based studies on management of long-chain FAO defects are lacking, we carried out a retrospective analysis of 75 patients from 18 metabolic centres in Germany, Switzerland, Austria and the Netherlands with special regard to treatment and disease outcome. Dietary treatment is effective in many patients and can prevent acute metabolic derangements and prevent or reverse severe long-term complications such as cardiomyopathy. However, 38% of patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency had intermittent muscle weakness and pain despite adhering to therapy. Seventy-six per cent of patients with disorders of the mitochondrial trifunctional protein (TFP)-complex including long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, had long-term myopathic symptoms. Of these, 21% had irreversible peripheral neuropathy and 43% had retinopathy. The main principle of treatment was a fat-reduced and fat-modified diet. Fat restriction differed among patients with different enzyme defects and was strictest in disorders of the TFP-complex. Patients with a medium-chain fat-based diet received supplementation of essential long-chain fatty acids. l-Carnitine was supplemented in about half of the patients, but in none of the patients with VLCAD deficiency identified by newborn screening. In summary, in this cohort the treatment regimen was adapted to the severity of the underlying enzyme defect and thus differed among the group of long-chain FAO defects.

Methionine adenosyltransferase (MAT) I/III deficiency with concurrent hyperhomocysteinaemia: two novel cases.

This study reports three novel mutations of the methionine adenosyltransferase (MAT) lA gene and confirms that hyperhomocysteinaemia may be a characteristic finding in MAT I/III deficiency. Thus, MAT I/III deficiency is important in the differential diagnoses of hyperhomocysteinaemia, which may lead to clinical complications of MAT I/III deficiency.

[Clinical and genetic results with reference to corneal alterations in Lowe-syndrome]. / Klinische und genetische Befunde unter besonderer Betrachtung kornealer Veränderungen bei Lowe-Syndrom.

BACKGROUND: The Lowe oculo-cerebro-renal syndrome (OCRL1) is a rare X-linked disease which causes impairment of visual acuity. The situation may be further complicated by corneal alterations. PATIENTS AND METHODS: In total seven patients from different families were clinically examined by slit-lamp examination, funduscopy, measurement of the intraocular pressure and ultrasound sonography. Molecular genetic analysis was performed in six patients by sequencing large PCR amplicons with a DNA sequencer and the ABI PRISM Sequence navigator software. RESULT: All affected boys were aphakic. Due to high intraocular pressure, iridectomy, goniotomy, cyclo-cryo treatment or trabeculectomy were performed. All patients showed opacity or pannus-like alterations of the cornea. Molecular genetic analysis revealed four novel and two known mutations. CONCLUSION: Reduced visual acuity was partly explained by morphological changes due to the underlying genetic defect and the development of cataract and glaucoma. Opacity of the cornea as well as alterations of the cornea seem to be a major problem in the course of the disease.

T-cell-depleted peripheral blood stem cell transplantation for alpha-mannosidosis.

Alpha-mannosidosis (alpha-mannosidosis) is a lysosomal storage disease characterized by accumulation of oligosaccharides in various tissues leading to symptoms such as coarse facial features, dysostosis multiplex, hearing disabilities, mental developmental delay and skeletal involvement (dysostosis multiplex). Without treatment, the severe infantile onset form of this autosomal recessive disease leads to progressive neurodegeneration and sometimes to early death. Stem cell transplantation has been shown to be an effective treatment. In the five patients published so far, correction of skeletal abnormalities and improvement of neuropsychological capabilities have been observed. We report the first patient who received a T-cell-depleted peripheral blood stem cell transplantation (PBSCT) for alpha-mannosidosis. The diagnosis of alpha-mannosidosis was made at the age of 14 months. At the age of 24 months, he underwent PBSCT with T-cell depletion by CD34-positive selection from his HLA phenotypically identical mother. Conditioning was carried out with busulfan (20 mg/kg), cyclophosphamide (200 mg/kg), OKT3 and methylprednisolone. The patient is alive and well 27 months after PBSCT and has made significant developmental progress. The pattern of urinary oligosaccharides has returned to almost normal. CD34-positive-selected PBSCT is a feasible option to reduce risk for GVHD for these patients.

Hepatic carnitine palmitoyltransferase I deficiency: acylcarnitine profiles in blood spots are highly specific.

BACKGROUND: In carnitine palmitoyltransferase I (CPT-I) deficiency (MIM 255120), free carnitine can be increased with no pathologic acylcarnitine species detectable. As inclusion of CPT-I deficiency in high-risk and newborn screening could prevent potentially life-threatening complications, we tested whether CPT-I deficiency might be diagnosed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). METHODS: A 3.2-mm spot of whole blood dried on filter paper was extracted with 150 microL of methanol. After derivatization of carnitine and acylcarnitines to their butyl esters, the samples were analyzed by ESI-MS/MS with 37.5 pmol of L-[(2)H(3)]carnitine and 7.5 pmol of L-[(2)H(3)]palmitoylcarnitine as internal standards. RESULTS: In all dried-blood specimens from each of three patients with CPT-I deficiency, we found an invariably increased ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)]. The ratio in patients was between 175 and 2000, or 5- to 60-fold higher than the ratio for the 99.9th centile of the normal newborn population in Bavaria (n = 177 842). No overlap with the values of children that were known to be supplemented with carnitine was detected [C0/(C16 + C18), 34 +/- 30; mean +/- SD; n = 27]. CONCLUSIONS: ESI-MS/MS provides a highly specific acylcarnitine profile from dried-blood samples. The ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)] is highly specific for CPT-I deficiency and may allow presymptomatic diagnosis.

Cloning of the human MCCA and MCCB genes and mutations therein reveal the molecular cause of 3-methylcrotonyl-CoA: carboxylase deficiency.

3-Methylcrotonyl-CoA: carboxylase (EC 6.4.1.4; MCC) deficiency is an inborn error of the leucine degradation pathway (MIM *210200) characterized by increased urinary excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. The clinical phenotypes are highly variable ranging from asymptomatic to profound metabolic acidosis and death in infancy. Sequence similarity with Glycine max and Arabidopsis thaliana genes encoding the two subunits of MCC permitted us to clone the cDNAs encoding the alpha- and beta-subunits of human MCC. The 2580 bp MCCA cDNA encodes the 725 amino acid biotin-containing alpha-subunit. The MCCA gene is located on chromosome 3q26-q28 and consists of 19 exons. The 2304 bp MCCB cDNA encodes the non-biotin-containing beta-subunit of 563 amino acids. The MCCB gene is located on chromosome 5q13 and consists of 17 exons. We have sequenced both genes in four patients with isolated biotin-unresponsive deficiency of MCC. In two of them we found mutations in the MCCA gene. Compound heterozygosity for a missense mutation (S535F) and a nonsense mutation (V694X) were identified in one patient. One heterozygous mutation (S535F) was found in another patient. The remaining two patients had mutations in the MCCB gene. One consanguineous patient was homozygous for a missense mutation (R268T). In the other we identified a missense mutation in one allele (E99Q) and allelic loss of the other. Mutations were correlated with an almost total lack of enzyme activity in fibroblasts. These data provide evidence that human MCC deficiency is caused by mutations in either the MCCA or MCCB gene.

Molecular analysis and long-term follow-up of patients with different forms of 6-pyruvoyl-tetrahydropterin synthase deficiency.

UNLABELLED: The outcome of 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, the most common form of tetrahydrobiopterin (BH4) deficiency, depends on factors such as severity of the disease, type of mutation, time of diagnosis, and mode of treatment. We investigated five patients from four different families, four of them presenting with the severe form of PTPS deficiency and one with the mild peripheral form. In this study, missense (L26F, T67M, P87L, V124L, D136G, D136V) and nonsense (R15-16ins) mutations were detected by reverse transcriptase polymerase chain reaction and sequence analysis. Two patients with the severe form were compound heterozygotes (T67M/P87L and D136G/R15-16ins), two siblings were homozygous for the D136V mutation, and in the patient with the mild form, heterozygous L26F/V124L mutations were present. Two patients are on combined therapy with L-dopa/carbidopa/5-hydroxytryptophan plus BH4, the siblings are on monotherapy with BH4, and the patient with the mild form is now off treatment, presenting with normal plasma phenylalanine levels. CONCLUSION: Long-term follow-up shows that the outcome of 6-pyruvoyl-tetrahydropterin synthase deficiency benefits from treatment started in the first months of life and that the phenotype may change with age. Additionally, depending on the type of mutations, prenatal damage to the fetus may multiply the clinical abnormalities and thus worsen the prognosis of the disease. In patients initially diagnosed with the mild peripheral form of the disease, therapy with tetrahydrobiopterin should be stopped after some time to test whether hyperphenylalaninaemia was only a transient condition.

Combined D-2- and L-2-hydroxyglutaric aciduria with neonatal onset encephalopathy: a third biochemical variant of 2-hydroxyglutaric aciduria?

Two distinct disorders with elevated urinary excretion of 2-hydroxyglutaric acid are known: L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria. This paper presents clinical and biochemical studies in three patients and unsuccessful prenatal diagnosis in one case with combined D-2- and L-2-hydroxyglutaric aciduria. We suggest that these patients, who displayed a phenotype of neonatal onset metabolic encephalopathy, present a third variant of 2-hydroxyglutaric aciduria. Prenatal diagnosis is not reliable in this disorder.

Carnitine-acylcarnitine translocase deficiency: metabolic consequences of an impaired mitochondrial carnitine cycle.

We describe a patient with carnitine-acylcarnitine translocase deficiency (MIM 212138), who presented with neonatal generalized seizures, heart failure, and coma. Laboratory evaluation revealed hypoglycemia, hyperammonemia, lactic acidemia, hyperuricemia, and mild dicarboxylic aciduria. The fact that total plasma carnitine (7.1 micromol/l [20-30]) and free carnitine (1.9 micromol/l [12-18]) were low together with a high acylcarnitine/free carnitine ratio of 2.7 [0.4-1.0] prompted acylcarnitine analysis. This revealed the presence of large amounts of long-chain derivatives including C(16:0), C(16:1), C(18:1), C(18:2). Based on these findings carnitine-acylcarnitine translocase deficiency was suspected which was confirmed by enzyme studies in fibroblasts. The underlying complex metabolic consequences of this defect are reviewed. Prenatal diagnosis was performed in a subsequent pregnancy and a defect ruled out by measurement of carnitine-acylcarnitine translocase activity in cultured chorionic villi cells. As the clinical recognition of a life-threatening fatty acid oxidation disorder may be difficult, defects in this pathway should be considered in any child with coma, an episode of a Reye-like syndrome, and cardiomyopathy. Since routine laboratory tests often do not provide clues about potential disorders and profiles of urinary organic acids may not be characteristic, we recommend to measure free carnitine and acylcarnitines in plasma in any child with hyperammonemia, hypo/hyperketotic hypoglycemia or lactic acidemia for prompt treatment, proper genetic counseling, and potential prenatal diagnosis.

[Model project for updating neonatal screening in Bavaria: concept and initial results]. / Modellprojekt zur Neuordnung des Neugeborenen-Screenings in Bayern: Konzeption und erste Ergebnisse.

The newborn screening programme in Bavaria was confronted with several problems. Number of disorders and process quality no longer complied with screening guidelines. Mixed financing, distributed between the state (PKU, galactosaemia) and health insurances (hypothyroidism) had promoted an increasing dissipation of the system. Notified participation rates had dropped to < 80%. Increasing need for a second screening due to early discharge was an additional challenge. To overcome these problems, and considering the availability of improved screening methodology (tandem mass spectrometry) the programme was reorganised. The project, which started on Jan 1, 1999, is based on a cooperation model between laboratory (logistics, analysis), universities (treatment, scientific evaluation), and public health services (coordination, tracking). Time of blood sampling was predated to the third day of life. Screening was extended to biotinidase deficiency, congenital adrenal hyperplasia (CAH) and by introduction of tandem mass spectrometry for screening of many other disorders (besides PKU). Insurances now finance complete laboratory analysis which was transferred to the private sector. To enable all newborn to participate, the names of screened children are matched against birth lists by public health services on a regional basis. Recalls and conspicuous results are consistently followed up until disorders are either excluded or confirmed. Two clinical hotlines were established in the children's hospitals of the universities in Munich (Southern Bavaria) and in Erlangen (Northern Bavaria). Written consent is required for participation in the programme. Participation in the new programme could be continually increased; coverage is > 95% since April. In several cases screening was made up for not tested children by contacting their parents. Omitted screening was mostly due to misunderstandings regarding testing responsibility or lost samples. Altogether 52 cases of disorder were found in the 87,000 newborn screened until August 1999. Hence, the detection rate of children affected by inborn errors of metabolism was about twice as high than before changes. Among the newly screened diseases CAH was detected most often (11 cases). In 22 cases diagnosis was based on the use of tandem mass spectrometry. Among these (besides PKU, 9 cases) MCAD deficiency (6 cases) was detected most frequently. Whereas recall rates of most disorders were < 0.1%, screening for CAH still revealed a high recall rate, particularly in premature births. Second screening due to early discharge (< 48 h) was required in 1.3%. About 20% of pending recalls required contacting birth hospitals, doctors, midwives or parents. So far all affected children could be brought to treatment in time.

Characteristics of L-ornithine: 2-oxoacid aminotransferase and potential prenatal diagnosis of gyrate atrophy of the choroid and retina by first trimester chorionic villus sampling.

A deficiency of the mitochondrial matrix enzyme L-ornithine: 2-oxoacid aminotransferase causes gyrate atrophy of the choroid and retina with hyperornithinemia (MIM 258870), a blinding degenerative disease, which is inherited as an autosomal recessive trait. We have developed a sensitive microradioisotopic method for enzyme assay by using 2-oxo-[5-14C] glutarate as the substrate and performing the separation of the product, [5-14C] glutamate from the substrate on a cation-exchange column. The enzyme activity was determined in human and rat tissues and in cultured cells. The enzyme activity in fibroblasts from a patient was deficient and that of the parents ranged between 25 and 60% of the control values. In addition we have found the enzyme expressed in native and cultured chorionic villi indicating a potential detection of the disease during the first trimester of pregnancy.

Carrier assessment in families with lowe oculocerebrorenal syndrome: novel mutations in the OCRL1 gene and correlation of direct DNA diagnosis with ocular examination.

Lowe oculocerebrorenal syndrome (OCRL) (MIM 309000) is a rare X-linked multisystem disorder characterized by congenital cataracts, muscular hypotonia, areflexia, mental retardation, maladaptive behavior, renal tubular dysfunction, vitamin-D-resistant rickets, and scoliosis. The underlying gene OCRL1 is located on chromosome Xq25-q26 and contains 24 exons. It encodes a 105-kDa phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) 5-phosphatase that is localized to the Golgi complex. To confirm the clinical diagnosis and to assess the carrier state of female relatives for genetic counseling we examined 6 independent patients and their families (a total of 23 individuals) using an improved mutation screening strategy for the OCRL1 gene by sequencing of large PCR amplicons. Four novel and two known mutations were identified: three premature terminations caused by either frameshift mutations (1899insT in exon 17 and 2104-2105delGT in exon 18) or a nonsense mutation (1399C > T in exon 12), two missense mutations (1676G > A and 1754C > T in exon 15), and a 6-bp deletion (1609-1614delAAGTAT in exon 14). An ophthalmological examination was performed in all patients and 14 female relatives. All genotypically proven carrier females showed characteristic lenticular opacities, while all proven noncarriers were lacking this phenotypic finding. The results confirm that ophthalmological evaluation is an apparently reliable first-line method to ascertain the carrier state in Lowe oculocerebrorenal syndrome. The high expressivity of lenticular symptoms in OCRL1 gene carriers is consistent with the hypothesis that (PtdIns[4,5]P(2)) 5-phosphatase activity has low functional reserve capacity for maintaining a balanced homeostasis of lenticular metabolism.

[Dorfman-Chanarin syndrome--a neutral lipid storage disease]. / Dorfman-Chanarin-Syndrom--Eine Neutrallipidspeicherkrankheit.

Dorfman-Chanarin syndrome is a rare, autosomal recessive inherited lipid storage disease with skin manifestations in form of congenital ichthyotic erythroderma. Demonstration of lipid vacuoles in neutrophils from peripherals blood smears in patients with ichthyotic erythroderma leads to the diagnosis. Other organ systems, such as CNS, liver, muscle, ears and eyes, are frequently involved. Since Dorfman-Chanarin syndrome may present with just skin findings, it should be included in the differential diagnosis of the congenital ichthyoses. Microscopic examination of peripheral blood smears is recommended in all patients with ichthyosis. We summarize the current data on the pathogenesis, diagnosis, differential diagnosis, and therapeutic options in Dorfman-Chanarin syndrome, give an overview of the clinical manifestations of the 23 patients affected with this rare disease.

An unusual presentation of medium-chain acyl coenzyme A dehydrogenase deficiency.

OBJECTIVE: To report an atypical presentation of medium-chain acyl Coenzyme A dehydrogenase deficiency in a 13-year-old girl with hyperammonemic encephalopathy and orotic aciduria meeting the accepted criteria for diagnosis of a female heterozygous for ornithine transcarbamylase deficiency. DESIGN: Case report and definitive biochemical testing. SETTING: Children's hospital and university laboratory. PARTICIPANT: One teenager. INTERVENTIONS: Diagnosis and treatment with carnitine. MEASUREMENTS/MAIN RESULTS: Assay ornithine transcarbamylase deficiency had normal results. The diagnosis was confirmed by DNA analysis, which revealed homozygosity for prevalent mutation (the adenine to guanine transition at position 985). CONCLUSIONS: Patients with a clinical diagnosis of Reye's syndrome have, in general, an inborn error of metabolism. Medium-chain acyl Coenzyme A dehydrogenase deficiency and other disorders of fatty acid oxidation may present long after infancy. They may mimic the presentation of defects in the urea cycle.