False-positive results in neonatal screening for cystic fibrosis based on a three-stage protocol (IRT/DNA/IRT): Should we adjust IRT cut-off to ethnic origin?

Since 1979, newborn screening for cystic fibrosis (CF) has been possible by measuring immunoreactive tryspinogen (IRT) in blood spots. In France, a programme based on a three-stage strategy (IRT/DNA/IRT) started in 2002. In the Rhône-Alpes area, the positive screening rate (i.e. the proportion of samples sent for genotyping) observed after the first IRT measurement was higher than the expected rate (0.65% versus 0.50%), without a greater CF incidence. We hypothesized that the IRT reference range could differ according to the ethnic origin of the newborns. 35 141 newborns were studied and divided into two groups: European ethnic group 26 324 (75%) and North African ethnic group 8817 (25%). 243 positive newborns were identified: 146 (60%) in the European ethnic group and 97 (40%) in the North African ethnic group. Three CF patients and 11 unaffected heterozygotes were found in the European group, but no mutations were found in the North African group. Mean IRT values and the percentage of IRT values over the cut-off were significantly higher in the North African group than in the European group (mean IRT = 21.17 microg/L and 19.74 microg/L, p < 0.0001; %IRT > cut-off = 1.1% and 0.5%, respectively). For the positive screened newborns, term and IRT mean were comparable, whereas birth weight was higher in the North African ethnic group. These results lead us to conclude that (i) newborns from families of North African origin have higher IRT values and (ii) most of the positive screened newborns in this population could be considered as 'false positives'. These conclusions could explain, in part, the large variations seen in the positive screening rate in the French CF neonatal screening and raise the question whether it is relevant to adapt cut-off to ethnic origin of the newborns.

[Application of tandem mass spectrometry to neonatal screening of inherited metabolic diseases: focus on present developments]. / La spectrométrie de masse en tandem appliquée au dépistage néonatal des maladies héréditaires du métabolisme: le point sur les utilisations actuelles.

The recent evolution of tandem mass spectrometry allows to diagnose more than twenty inherited metabolic diseases within a single blood spot. Nowadays, it is technically possible to screen newborns for most of fatty acid oxidation, organic acid and amino acid disorders. An important number of prospective pilot studies, using tandem mass spectrometry, have been done worldwide. However, several technical, economical, medical and ethical problems are raised by these applications. This review is intended to focus on this technology and to resume results from the main international studies.

CDG IIx with unusual phenotype.

Congenital disorders of glycosylation (CDG) are a group of genetic diseases characterized by defective protein glycosylation. N-glycosylation defects are divided into two groups (I and II). CDG group II (types IIa to IIe) refers to defects in the Golgi processing of protein-bound glycans. We report a patient with CDG IIx and an unusual phenotype.

Outcome of 27 patients with Hurler's syndrome transplanted from either related or unrelated haematopoietic stem cell sources.

Over the last 15 years, we have performed a total of 30 haematopoietic stem cell transplants on 27 children suffering from Hurler's syndrome. These children were of median age 11 months at the time of diagnosis and 25 months at the time of transplantation. The phenotype was severe in 21 cases (78%). The donor was familial in 13 cases: nine genotypically identical, one phenotypically identical father and three HLA-mismatched donors. Unrelated donors were selected in 17 cases: four phenotypically identical and 13 with 1-4 HLA mismatches. The conditioning regimen generally consisted of busulphan 600 mg/m(2) plus cyclophosphamide (Endoxan) 260 mg/kg and cyclosporin with methotrexate for GvHD prophylaxis. Rabbit anti-thymocyte globulin (Thymoglobuline) was given for all unrelated or familial mismatched transplantations. The median nucleated cell dose infused was 6.00 x 10(8) TNC/kg. No bone marrow (apart from one) was T cell depleted. For first transplants, engraftment was observed in 23/27 patients (pts) (85%). Primary graft failure was observed in 4/27 patients (16%), two were retransplanted from an unrelated donor, one with success. Four patients have died. The primary cause of death was infection in three cases (TRM : 11%) and disease progression in one case, after primary graft failure. Of the 23 living patients, two have disease progression after graft failure and 21 (78%) have functional grafts with a favourable long-term outcome after a median follow-up of 4.7 years, having either full or mixed chimaerism. Among surviving patients with functional grafts, 13 (62%) were transplanted from unrelated donors of whom 10 (77 %) had HLA disparities. There was a remarkably low incidence of GvHD. In our experience, haematopoietic stem cell transplantation using an HLA-matched familial donor or an HLA-matched or -mismatched unrelated donor without T cell depletion or irradiation can achieve a favourable outcome in Hurler's syndrome, with improved cognitive function, but with a limited effect on the corneas and skeleton.

Prenatal diagnosis and carrier detection for molybdenum cofactor deficiency type A in northern Israel using polymorphic DNA markers.

Molybdenum cofactor deficiency (MoCoD) is an autosomal recessive, fatal neurological disorder, characterized by the combined deficiency of sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase. We have recently reported an excessive occurrence of this fatal disorder among segments of the Arab population in Northern Israel suggesting that the true incidence of MoCoD is probably underestimated in this highly inbred population. This lethal disease can be diagnosed prenatally by assay of sulphite oxidase activity in chorionic villus samples in pregnancies of couples who have had previously affected children (obligatory carriers). However, to date, there is no biochemical assay for carrier detection among the population at risk. Recently we demonstrated the linkage of a MoCoD gene to an 8-cM region on chromosome 6p21.3 in two consanguineous Israeli-Arab unrelated kindreds. The description of the MOCS1 gene that maps to the same region and which carries multiple mutations in MoCoD type A followed this finding. We describe here one additional kindred of Arab-Israeli origin, which is also linked to the MOCS1 locus, and demonstrate the feasibility of prenatal diagnosis and carrier detection using microsatellite markers in selected families when mutations are unknown. A complete correlation between the biochemical and DNA assays was found in a total of six samples (five chorionic villus and one amniocyte culture sample) obtained from the three MoCoD families.

Dietary therapy in two patients with a mild form of sulphite oxidase deficiency. Evidence for clinical and biological improvement.

We report an attempt at dietetic therapy in two unrelated patients with isolated sulphite oxidase deficiency, with a mild clinical course and late onset of symptoms. In case 1, disease started at 15 months with an acute crisis of agitation, unexplained crying and restlessness following otitis. Case 2 was diagnosed at 10 months when she presented with slight motor delay and dislocation of lenses. In both cases, sulphite oxidase activity measured in fibroblasts was undetectable. Therapy consisted of a diet low in protein from natural foods (daily methionine intake 130-150 mg) and a synthetic amino acid mixture (50 g per day) without cystine and methionine (Xmet, Cys Maxamaid, SHS International Ltd). A comparison of clinical and biochemical parameters was made between the period before treatment and after 2 years of treatment. Restriction in protein and sulphur amino acids brought about a dramatic decrease of urinary thiosulphate and S-sulphocysteine. It also brought about a generalized hypoaminoacidaemia with a low plasma methionine and cystine in both patients. Furthermore, both patients grew normally with no signs of neurological deterioration, and there was evidence of progress in psychomotor development.

Molybdenum cofactor deficiency: first prenatal genetic analysis.

Molybdenum cofactor (MoCo) deficiency leads to a combined deficiency of the molybdo-enzymes sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase. No therapy is known for this rare disease, which results in neonatal seizures and other neurological symptoms identical to sulphite oxidase deficiency. It is inherited autosomal-recessively and leads to early childhood death. Prenatal diagnosis has been performed since 1983 by the measurement of sulphite oxidase activity, but no enzymatic carrier diagnosis is possible. The human genes necessary for MoCo biosynthesis have recently been cloned and mutations in the bicistronic MOCS1 gene could be identified in most European patients. In a Danish family we have now performed enzymatic and molecular genetic analysis in parallel after chorionic villus sampling. The sulphite oxidase activity in uncultured CVS material was found to be normal. A MOCS1 splice site mutation, found homozygous in the affected patient, was found in a heterozygous state in cultured chorionic cells. This confirmed that the fetus was not affected, since heterozygous carriers of a MoCo deficiency allele do not display any symptoms.

Human molybdopterin synthase gene: genomic structure and mutations in molybdenum cofactor deficiency type B.

Biosynthesis of the molybdenum cofactor (MoCo) can be divided into (1) the formation of a precursor and (2) the latter's subsequent conversion, by molybdopterin synthase, into the organic moiety of MoCo. These two steps are reflected by the complementation groups A and B and the two formally distinguished types of MoCo deficiency that have an identical phenotype. Both types of MoCo deficiency result in a pleiotropic loss of all molybdoenzyme activities and cause severe neurological damage. MOCS1 is defective in patients with group A deficiency and has been shown to encode two enzymes for early synthesis via a bicistronic transcript with two consecutive open reading frames (ORFs). MOCS2 encodes the small and large subunits of molybdopterin synthase via a single transcript with two overlapping reading frames. This gene was mapped to 5q and comprises seven exons. The coding sequence and all splice site-junction sequences were screened for mutations, in MoCo-deficient patients in whom a previous search for MOCS1 mutations had been negative. In seven of the eight patients whom we investigated, we identified MOCS2 mutations that, by their nature, are most likely responsible for the deficiency. Three different frameshift mutations were observed, with one of them found on 7 of 14 identified alleles. Furthermore, a start-codon mutation and a missense mutation of a highly conserved amino acid residue were found. The locations of the mutations confirm the functional role of both ORFs. One of the patients with identified MOCS2 mutations had been classified as type B, in complementation studies. These findings support the hypothetical mechanism, for both forms of MoCo deficiency, that formerly had been established by cell-culture experiments.

Mutations in a polycistronic nuclear gene associated with molybdenum cofactor deficiency.

All molybdoenzymes other than nitrogenase require molybdopterin as a metal-binding cofactor. Several genes necessary for the synthesis of the molybdenum cofactor (MoCo) have been characterized in bacteria and plants. The proteins encoded by the Escherichia coli genes moaA and moaC catalyse the first steps in MoCo synthesis. The human homologues of these genes are therefore candidate genes for molybdenum cofactor deficiency, a rare and fatal disease. Using oligonucleotides complementary to a conserved region in the moaA gene, we have isolated a human cDNA derived from liver mRNA. This transcript contains an open reading frame (ORF) encoding the human moaA homologue and a second ORF encoding a human moaC homologue. Mutations can be found in the majority of MoCo-deficient patients that confirm the functional role of both ORFs in the corresponding gene MOCS1 (for 'molybdenum cofactor synthesis-step 1'). Northern-blot analysis detected only full-length transcripts containing both consecutive ORFs in various human tissues. The mRNA structure suggests a translation reinitiation mechanism for the second ORF. These data indicate the existence of a eukaryotic mRNA, which as a single and uniform transcript guides the synthesis of two different enzymatic polypeptides with disease-causing potential.

Localization of a gene for molybdenum cofactor deficiency, on the short arm of chromosome 6, by homozygosity mapping.

Molybdenum cofactor deficiency (MoCoD) is a fatal disorder manifesting, shortly after birth, with profound neurological abnormalities, mental retardation, and severe seizures unresponsive to any therapy. The disease is a monogenic, autosomal recessive disorder, and the existence of at least two complementation groups suggests genetic heterogeneity. In humans, MoCoD leads to the combined deficient activities of sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. By using homozygosity mapping and two consanguineous affected kindreds of Israeli-Arab origin, including five patients, we demonstrated linkage of a MoCoD gene to an 8-cM region on chromosome 6p21.3, between markers D6S1641 and D6S1672. Linkage analysis generated the highest combined LOD-score value, 3.6, at a recombination fraction of 0, with marker D6S1575. These results now can be used to perform prenatal diagnosis with microsatellite markers. They also provide the only tool for carrier detection of this fatal disorder.

Genomic structure and mutational spectrum of the bicistronic MOCS1 gene defective in molybdenum cofactor deficiency type A.

Molybdenum cofactor (MoCo) deficiency is a rare and devastating disease resulting in neonatal seizures and other neurological symptoms identical to those of sulphite oxidase deficiency. It is an autosomal recessive disease and no therapy is known. Most patients harbour MOCS1 mutations, which are found in both open reading frames of this unusual gene encoding the first two enzymes required in the MoCo biosynthesis pathway, MOCS1 A and MOCS1 B, in a single transcript. We describe genomic details as a prerequisite for comprehensive mutation analysis. In an initial cohort of 24 MoCo deficiency patients, we identified 13 different mutations on 34 chromosomes, with a mutation detection rate of 70%. Five mutations were observed in more than one patient and together accounted for two thirds of detected mutations. These comprise the most frequent mutation, R319Q, which is restricted to England, two Danish/German mutations (one missense and one splice site mutation), a missense mutation found in England and Germany, and a "Mediterranean" frameshift mutation. All patients with identified mutations are either homozygous or compound heterozygous for mutations in either of the two open reading frames corresponding to MOCS1 A and MOCS1 B, respectively. This observation suggests the existence of more than the two previously described complementation groups in MoCo biosynthesis.

Spherophakia associated with molybdenum cofactor deficiency.

Molybdenum cofactor deficiency is an autosomal recessive disorder characterized by lack of activity of the enzymes sulfite oxidase, aldehyde oxidase, and xanthine dehydrogenase or oxidase. The clinical manifestations are indistinguishable from those of isolated sulfite oxidase deficiency: craniofacial alterations, intractable neonatal convulsions, very severe mental retardation, lens dislocation, and death in the first decade of life. Lens dislocation is found in nearly all patients after neonatal age. In the present case it developed late (at the age of 8 years) and was preceded by bilateral spherophakia. We hypothesize that an abnormal relaxation of the zonular fibers is the cause of spherophakia in this disease; this causes lens dislocation eventually, after days, months, or years.

A mild form of infantile isolated sulphite oxidase deficiency.

Neonatal sulphite oxidase deficiency is characterised by severe neurologic dysfunction, brain atrophy, dislocation of the lens and increased urinary excretion of sulphite, thiosulphate, taurine and S-sulphocysteine, and by a low plasma cystine. We present clinical, neuroradiological and biochemical data of a patient with late onset symptoms comparing this presentation with the neonatal form and stressing the difficulties of the diagnosis of this disorder.

[Sulfite oxidase deficiency presenting as Leigh syndrome]. / Déficit en sulfite oxydase se présentant comme un syndrome de Leigh.

BACKGROUND: An enzyme deficiency can be demonstrated in 15 to 20% of cases of Leigh syndrome. A case of isolated sulphite oxidase deficiency is reported in a girl presenting with Leigh syndrome. CASE REPORT: An 8 month-old girl was admitted suffering from hypotonia and slow increase of head circumference (-1 SD). Examination showed spastic quadriplegia, dyskinesia, axial hypotonia and difficulties in swallowing. The patient had a coarse face, broad nasal bridge, long philtrum and ectopia lentis. Brain CT scan showed bilateral hypodensity of lenticular nuclei and moderate cortical atrophy. Amino acid chromatography showed accumulation of S sulfocysteine and low levels of cysteine. The sulphite test was positive. Sulphite oxidase activity in fibroblasts and liver was undetectable contrasting with a normal activity of xanthine oxidase. Progressive brain damage led to death at 1 year of age. Prenatal diagnosis of sulphite oxidase deficiency was made in two further pregnancies. CONCLUSIONS: The search for sulphite oxidase deficiency must be included in discussing the etiology of Leigh syndrome; the sulphite test is a simple method of screening such cases.

Molybdenum cofactor deficiency in two siblings: diagnostic difficulties.

Two siblings with molybdenum cofactor deficiency are presented. They showed clinical, biochemical and neuroradiological features very similar to those of the few previously described cases. Difficulties in diagnosis are emphasised.