High rate of autonomic neuropathy in Cornelia de Lange Syndrome.

BACKGROUND: Cornelia de Lange Syndrome (CdLS) is a rare congenital disorder characterized by typical facial features, growth failure, limb abnormalities, and gastroesophageal dysfunction that may be caused by mutations in several genes that disrupt gene regulation early in development. Symptoms in individuals with CdLS suggest that the peripheral nervous system (PNS) is involved, yet there is little direct evidence. METHOD: Somatic nervous system was evaluated by conventional motor and sensory nerve conduction studies and autonomic nervous system by heart rate variability, sympathetic skin response and sudomotor testing. CdLS Clinical Score and genetic studies were also obtained. RESULTS: Sympathetic skin response and sudomotor test were pathological in 35% and 34% of the individuals with CdLS, respectively. Nevertheless, normal values in large fiber nerve function studies. CONCLUSIONS: Autonomic nervous system (ANS) dysfunction is found in many individuals with Cornelia de Lange Syndrome, and could be related to premature aging.

Disruption of NIPBL/Scc2 in Cornelia de Lange Syndrome provokes cohesin genome-wide redistribution with an impact in the transcriptome.

Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF.

Expanding the clinical spectrum of the 'HDAC8-phenotype' - implications for molecular diagnostics, counseling and risk prediction.

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous disorder characterized by typical facial dysmorphism, cognitive impairment and multiple congenital anomalies. Approximately 75% of patients carry a variant in one of the five cohesin-related genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Herein we report on the clinical and molecular characterization of 11 patients carrying 10 distinct variants in HDAC8. Given the high number of variants identified so far, we advise sequencing of HDAC8 as an indispensable part of the routine molecular diagnostic for patients with CdLS or CdLS-overlapping features. The phenotype of our patients is very broad, whereas males tend to be more severely affected than females, who instead often present with less canonical CdLS features. The extensive clinical variability observed in the heterozygous females might be at least partially associated with a completely skewed X-inactivation, observed in seven out of eight female patients. Our cohort also includes two affected siblings whose unaffected mother was found to be mosaic for the causative mutation inherited to both affected children. This further supports the urgent need for an integration of highly sensitive sequencing technology to allow an appropriate molecular diagnostic, genetic counseling and risk prediction.

Could a patient with SMC1A duplication be classified as a human cohesinopathy?

The disorders caused by mutations in genes encoding subunits and accessory proteins of cohesin complex are collectively termed as cohesinopathies. The best known cohesinopathy is Cornelia de Lange Syndrome (CdLS), which is a multisystem developmental disorder characterized by facial dysmorphism, limb malformations, growth and cognitive impairment. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), are responsible for ∼ 70% of CdLS cases. We describe a 16-year-old boy with facial dysmorphism, growth retardation, intellectual disability, hirsutism and small hands, who has a small Supernumerary Marker Chromosome (sSMC) present in mosaic form. sSMC is composed of two duplicated segments encompassing 17 genes including SMC1A gene, at the regions Xp11.22 and Xp11.21q11.1. Clinical comparison between our patient with a previously reported individual with a SMC1A duplication and four male carriers of similar sSMC reported in databases, suggest that they all share clinical features related to cohesinopathies. Although our patient does not have the classical CdLS craniofacial phenotype, he has pre and postnatal growth retardation, intellectual disability and mild musculoskeletal anomalies, features commonly seen in patients with cohesinopathies.

Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment.

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

A single-residue mutation, G203E, causes 3-hydroxy-3-methylglutaric aciduria by occluding the substrate channel in the 3D structural model of HMG-CoA lyase.

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.

Refining the diagnosis of mitochondrial HMG-CoA synthase deficiency.

Mitochondrial HMG-CoA synthase deficiency is an inherited metabolic disorder caused by a defect in the enzyme that regulates the formation of ketone bodies. Patients present with hypoketotic hypoglycaemia, encephalopathy and hepatomegaly, usually precipitated by an intercurrent infection or prolonged fasting. The diagnosis may easily be missed as previously reported results of routine metabolic investigations, urinary organic acids and plasma acylcarnitines may be nonspecific or normal, and a high index of suspicion is required to proceed to further confirmatory tests. We describe a further acute case in which the combination of urinary organic acids, low free carnitine and changes in the plasma acylcarnitine profile on carnitine supplementation were very suggestive of a defect in ketone synthesis. The diagnosis of mitochondrial HMG-CoA synthase deficiency was confirmed on genotyping, revealing two novel mutations: c.614G > A (R188H) and c.971T > C (M307T). A further sibling, in whom the diagnosis had not been made acutely, was also found to be affected. The possible effects of these mutations on enzyme activity are discussed.

Preperitoneal repair for recurrent inguinal hernia: laparoscopic and open approach.

BACKGROUND: The aim of this study was to investigate the outcome of preperitoneal repair using laparoscopic (TEP) and open (OPM) approach in recurrent inguinal hernia. METHODS: We performed a prospective controlled nonrandomized clinical study in 188 patients with 207 recurrent inguinal hernias over a period of 5 years. TEP repair was employed for 86 repairs, and OPM was used in 121 procedures. The main outcome measurements were: recurrence rate, operating time, hospital stay, and postoperative complications. RESULTS: There were three recurrences (1.7%). Two in the OPM group (1.8%) and one (1.3%) in the TEP group [ P=NS (not significant)]. The TEP procedure was faster than OPM for unilateral repair (40.8 vs 46.3 min) (P<0.001). Postoperative complications were more frequent in the OPM group (23.9%) than the TEP group (13.9%) ( P=NS). Hospital stay was significantly shorter in the TEP group (1.2 vs 3.9 days) (P<0.001). CONCLUSIONS: Preperitoneal approach (open or laparoscopic) seems to be a good option in recurrent inguinal hernia when these procedures are done by experienced surgeons.

Molecular basis of 3-hydroxy-3-methylglutaric aciduria.

3-Hydroxy-3-methylglutaric aciduria is a human autosomal recessive metabolic disorder that usually appears within the first year of life. The causes of this aciduria are lethal mutations in the gene encoding for 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL). HL is a mitochondrial matrix enzyme that catalyzes the last step of ketogenesis and leucine catabolism. This gene has been mapped to chromosome 1 at locus 1pter-p33 and its genomic organisation comprises 9 exons whose sizes vary between 64-678 bp. The human cDNA sequence was reported in 1993 with the first genetic study of two Acadian-French Canadian siblings. To date, 24 mutations in 36 patients have been described; most of them are single-base substitutions causing amino acid replacements and a variety of splicing defects. In the population studied two mutations appear predominant: g.122GA (8 patients and 15 alleles) frequent in Saudi Arabia, and g.109GT (6 patients and 12 alleles), prevalent in Spain. At least seven mutations are clustered in the second half of exon 2 affecting aminoacids E37, R41 and D42 and conforming a possible hot spot. The genotype-phenotype correlation is difficult to establish since the probands received different treatments, and the onset of an acute episode frequently depends on external factors such as fasting or acute illness.

Tumor estromal extragastrointestinal. Peculiaridades en su semiología y terapéutica clínica / Extragastrointestinal stromal tumor. Semiology and clinical therapy peculiarities

Los tumores estromales gastrointestinales con localización extragastrointestinal son muy infrecuentes. Suelen diagnosticarse cuando presentan un gran tamaño. Su naturaleza benigna o maligna es difícil de determinar. Los mejores parámetros histológicos para evaluar su pronóstico son: un alto grado de celularidad, la presencia de necrosis tumoral y poseer más de dos mitosis por cincuenta campos. Presentamos el caso de una paciente asintomática que, por un control ecográfico rutinario por hepatopatía crónica por virus C, fue diagnosticada de un tumor mesentérico. La enferma fue tratada quirúrgicamente. El estudio inmunohistológico de la tumoración confirmó un tumor estromal gastrointestinal. El análisis del caso presentado y la bibliografía consultada sugieren unos hallazgos clínicos propios de esta entidad. Se revisa y actualiza la histogénesis de estas neoplasias y se destaca la utilidad de nuevos fármacos para el control de la progresión tumoral (AU)

Extra-gastrointestinal stromal tumour--semiology and clinical therapy peculiarities.

The gastrointestinal stromal tumor with extragastrointestinal location are very infrequent. We often diagnose them when they show a big size. Their bening or malignant nature is difficult to fix. The best histological parameters to evaluate their prognosis are a high cellularity, the tumor-like necrosis presence and having more than two mitosis per fifty high-power fields. We introduce an asyntomatic patient's case by a routine echographical control for chronic hepatitis by C virus, that has been diagnosed of a mesentery tumor. The patient has been treated surgically. The inmunohistological study of the tumor had confirmed a stromal gastrointestinal tumor. The showed case's analysis and the considered bibliography suggest some clinical discoveries characteristic of this entity. The histogenesis of these neoplasias are examined and made up to date and the usefulness of the new medication to control check the tumor-like progress is emphasized.

Acetaminophen induces apoptosis of C6 glioma cells by activating the c-Jun NH(2)-terminal protein kinase-related cell death pathway.

Acetaminophen (AAP), a widely used analgesic drug, can damage various organs when taken in large doses. In this study, we investigate whether AAP causes cell damage by altering the early signaling pathways associated with cell death and survival. AAP caused time- and concentration-dependent apoptosis and DNA fragmentation of C6 glioma cells used as a model. AAP activated c-Jun N-terminal protein kinase (JNK) by 5.3-fold within 15 min. The elevated JNK activity persisted for up to 4 h before it returned to the basal level at 8 h. In contrast, activities of other mitogen-activated protein (MAP) kinases and the level of Akt phosphorylation in the cell survival pathway remained unchanged throughout the treatment. Wortmannin, an inhibitor of phosphatidylinositol-3 kinase, or SB203580, an inhibitor of p38 MAP kinase, did not reduce AAP-induced toxicity, indicating that these enzymes do not play a major role in cell toxicity. AAP-induced apoptosis was preceded by the sequential elevation of the pro-apoptotic Bax protein, cytochrome c release, and caspase-3 activity. Treatment with caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) significantly reduced AAP-induced caspase-3 activation and cytotoxicity. Transfection of cDNA for the dominant-negative mutant JNK-KR or stress-activated protein kinase kinase-1 Lys-->Arg mutant (SEK1-KR), an immediate upstream kinase of JNK, significantly reduced AAP-induced JNK activation and cell death rate. The noncytotoxic analog of AAP, 3-hydroxyacetanilide, neither increased JNK activity nor caused apoptosis. Pretreatment with YH439, an inhibitor of CYP2E1 gene transcription, markedly reduced CYP2E1 mRNA, protein content, and activity, as well as the rate of AAP-induced JNK activation and cell death. These data indicate that AAP can cause cell damage by activating the JNK-related cell death pathway, providing a new mechanism for AAP-induced cytotoxicity.

Genetic basis of mitochondrial HMG-CoA synthase deficiency.

Deficiency of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGS) is a recessive disorder of ketogenesis that has been previously diagnosed in two children with hypoglycaemic hypoketotic coma during fasting periods. Here, we report the results of molecular investigations in a third patient affected by this disease. Sequencing of the entire coding region of the HMGCS2 gene revealed two missense mutations, G212R and R500H. Mendelian inheritance was confirmed by the analysis of parental samples and neither of the mutations was found on 200 control chromosomes. Functional relevance was confirmed by in vitro expression studies in cytosolic HMGS-deficient cells. Whereas wild-type cDNA of the HMGCS2 gene reverted the auxotrophy for mevalonate, the cDNAs of the mutants did not. The disease may be recognised by specific clinical and biochemical features but it is difficult to confirm enzymatically since the gene is expressed only in liver and testis. Molecular studies may facilitate or confirm future diagnoses in affected patients.

Apoptosis of PC12 cells by 4-hydroxy-2-nonenal is mediated through selective activation of the c-Jun N-terminal protein kinase pathway.

Cytotoxic lipid peroxides such as 4-hydroxy-2-nonenal (HNE) are produced when cells are exposed to toxic chemicals. However, the mechanism by which HNE induces cell death has been poorly understood. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in PC12 cells by measuring the activities of the mitogen-activated protein (MAP) kinases involved in early signal transduction pathways. Within 15-30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before returning to control level after 1 h post-treatment. In contrast, activities of extracellular signal regulated kinase (ERK) and p38 MAP kinase remained unchanged from their basal levels. SEK1, an upstream kinase of JNK, was also activated (phosphorylated) within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 was demonstrated by the transient transfection of cDNA for wild type SEK1 and JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when the dominant negative mutant of SEK1 was co-transfected with JNK. Pretreatment of PC12 cells with a survival promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, caused neither apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the HNE-mediated apoptosis of PC12 cells is likely to be mediated through the selective activation of the SEK1-JNK pathway without activation of ERK or p38 MAP kinase.

5-methoxytryptophol preserves hepatic microsomal membrane fluidity during oxidative stress.

Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation.

The pharmacokinetic characteristics of glycolated humanized anti-Tac Fabs are determined by their isoelectric points.

To evaluate a method for preventing the nephrotoxicity caused by the high renal accumulation of radiolabeled or toxin-conjugated small immunoproteins used for cancer therapy, we conjugated humanized anti-Tac Fab fragments with various numbers of glycolate molecules [glycolated Fab fragments (glyco-Fabs)] and separated the conjugates by means of ion-exchange columns into three fractions, depending on their isoelectric points (pIs). We evaluated the biodistribution, pharmacokinetics, and catabolism in normal nude mice of nonglycolated Fab (pI > or = 9.3) and three different preparations of glyco-Fab, including strongly anionic glyco-Fab (sa-glyco-Fab: pI < or = 4.5), mildly anionic glyco-Fab (pI = 4.5-7), and mildly cationic glyco-Fab (pI = 7-9.3). In addition, the biodistributions of 125I-labeled sa-glyco-Fab and 131I-labeled nonglycolated Fab were evaluated in normal nude mice coinjected with 50 mg of L-lysine and/or 1 microg of furosemide and in a control group without coinjection. We then evaluated the serial biodistribution of 125I-labeled sa-glyco-Fab (4 microCi/1 microg) and 131I-labeled nonglycolated Fab (5 microCi/1 microg) in Tac antigen-positive (ATAC4) and -negative (A431) tumor-bearing nude mice with s.c. tumor xenografts derived from Tac antigen-positive ATAC4 cells and receptor-negative A431 cells. These animals were coinjected with 30 mg of lysine i.v. and 30 mg of lysine i.p. 15 min after the radiolabeled Fab injection. To evaluate the biodistribution data and study scintigraphic imaging, we performed serial scintigraphy on normal and tumor-bearing mice with all four 131I-labeled preparations. 125I-labeled mildly cationic glyco-Fab and 131I-labeled nonglycolated Fab had similar distributions, except in the kidney. However, both 125I-labeled anionic glyco-Fab preparations showed significantly different distributions from both cationic Fabs in the blood, liver, lung, and spleen. Renal accumulation of all four radiolabeled Fab preparations increased significantly as the pI increased (P < 0.01). In addition, the intact fraction of Fab excreted into urine increased as pI decreased. Therefore, the glomerular filtration depended on whether the charge on the Fab was positive or negative. The proportion of Fab reabsorbed by the proximal tubules increased as pI increased. 125I-labeled sa-glyco-Fab and 125I-labeled mildly anionic glyco-Fab showed a similar distribution in the blood and all organs except the kidney. Lysine led to an additional blocking effect on proximal tubular uptake of both sa-glyco-Fab and nonglycolated Fab. Addition of furosemide yielded only a small effect when used with lysine. With lysine, the sa-glyco-Fab:nonglycolated Fab estimated integral radioactivity ratios were 4.7 and 0.7 in the ATAC4 tumor and in the kidney, respectively. The use of anionic fragments, which may be used in conjunction with lysine, represents a promising approach that may help decrease the renal toxicity of other small fragments, the molecular weights of which range from Mr 40,000 to 70,000, and, thereby, allow higher doses of radiation to the tumor.

Role of pinoline and melatonin in stabilizing hepatic microsomal membranes against oxidative stress.

We investigated the influence of pinoline (0.01-1.5 mM) on microsomal membrane fluidity before and after rigidity was induced by oxidative stress. In addition, we tested the effect of pinoline in the presence of 1 mM melatonin. The fluidity in rat hepatic microsomes was monitored using fluorescence spectroscopy and it was compared to the inhibition of malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) production as a reflection of lipid peroxidation. Below 0.6 mM, pinoline inhibited membrane rigidity in a manner parallel to its inhibitory effect on MDA + 4-HDA formation. At concentrations between 1-1.5 mM, pinoline was less effective in stabilizing microsomal membranes than was predicted from its inhibition of lipid peroxidation. The addition of 1 mM melatonin enhanced the membrane-stabilizing activity of pinoline (0.01-0.6 mM). This cooperative effect was not observed for concentrations of pinoline between 1-1.5 mM. When pinoline was tested without induced oxidative damage, 1-1.5 mM pinoline maintained membrane fluidity at the same level as that recorded after induced lipid peroxidation. The results suggest that pinoline may be another pineal molecule that prevents membrane rigidity mediated by lipid peroxidation and this ability is enhanced by melatonin.

Two missense point mutations in different alleles in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene produce 3-hydroxy-3-methylglutaric aciduria in a French patient.

Two novel point mutations in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene were found in a French patient with double heterozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by reverse transcriptase-polymerase chain reaction of the mRNA using five different pairs of oligonucleotides produced no differences in the fragments amplified with respect to the control. Single-strand conformation polymorphism analysis showed that only one amplified fragment was different in the patient vs. control. Sequencing of the amplified fragments showed two missense point mutations, A698G and T788C, each of them mixed with the wild-type sequence. These mutations produced the changes H233R and L263P, leading to changes in the enzyme activity, which was largely abolished. The father and one brother of the proband were heterozygous for the L263P mutation and the mother and one daughter were heterozygous for the H233R mutation, which confirms the double-heterozygous character of the patient. Another sibling was free of the mutations. An enzymatic restriction analysis has been proposed to screen the occurrence of these two mutations in future patients.

A nonsense mutation in the exon 2 of the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene producing three mature mRNAs is the main cause of 3-hydroxy-3-methylglutaric aciduria in European Mediterranean patients.

3-Hydroxy-3-methylglutaric aciduria is a rare recessive monogenic disorder that affects ketogenesis and the catabolism of L-leucine. We report the biochemical and molecular characterization of a mutation in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene in four new probands, three Spanish and one Turkish, affected by 3-hydroxy-3-methylglutaric aciduria, all homozygous for the nonsense mutation Glu37Ter, which was reported by our group in two probands of Portuguese and Moroccan origin (15). In addition to the aberrant mRNAs found in the two previous probands, a novel species of mature HL mRNA was observed in the patients studied here, since a new cDNA, skipped in exons 2 and 3, was obtained from the mRNAs by reverse-transcription PCR (RT-PCR). Thus, three mRNA species were produced in aberrant splicings as a result of this nonsense mutation: (i) one of the expected size that contains the premature stop codon UAA, (ii) another with a deletion of 84 bp corresponding to the whole of exon 2, and (iii) a new species found now, with a deletion of 192 bp corresponding to skipping of the whole of exons 2 and 3, whose translation product led to the loss of seven amino acids in the leader peptide and 57 amino acids in the terminal domain of the mature enzyme. The association of a nonsense mutation with the skipping of the exon that contains it, plus the following exon, is an unusual finding not seen previously in HL deficiencies. The mutation described here shows the highest incidence (> 37%) of total HL deficiencies reported.