Detailed molecular characterization of a novel IDS exonic mutation associated with multiple pseudoexon activation.

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

Polymicrogyria with dysmorphic basal ganglia? Think tubulin!

Dominant mutations in TUBB2B have been reported in patients with polymicrogyria. We further explore the phenotype associated with mutations in TUBB2B. Twenty patients with polymicrogyria (five unilateral) were tested for mutations in TUBB2B by Sanger sequencing. We identified two novel de novo mutations, c.743C>T (p.Ala248Val) and c.1139G>T (p.Arg380Leu) in exon 4 of TUBB2B in three unrelated families. Brain magnetic resonance images showed polymicrogyria involving predominantly the perisylvian regions. In addition, there was a dysmorphic appearance of the basal ganglia, thin corpus callosum, enlargement of the ventricles, thinning of the white matter and hypoplasia of pons and cerebellar vermis. This combination of associated features was absent in all 17 patients with polymicrogyria in whom no mutation was identified. This report underlines that the association of polymicrogyria with thin or absent corpus callosum, dysmorphic basal ganglia, brainstem and vermis hypoplasia is highly likely to result from mutations in TUBB2B and provides further insight in how mutations in TUBB2B affect protein function.

Inborn oxidative phosphorylation defect as risk factor for propofol infusion syndrome.

Propofol is an anesthetic agent widely used for induction and maintenance of anesthesia, and sedation in children. Although generally considered as reliable and safe, administration of propofol can occasionally induce a potentially fatal complication known as propofol infusion syndrome (PRIS). Mitochondrial dysfunction has been implicated in the pathogenesis of PRIS. We report on an adult patient with Leber hereditary optic neuropathy (LHON) who developed PRIS. He was a carrier of the m.3460G>A mutation, one of the major three pathogenic point mutations associated with LHON. The propositus was blind and underwent propofol sedation after severe head injury. Five days after start of propofol infusion, the patient died. The activity of complex I of the oxidative phosphorylation (OXPHOS) system was severely deficient in skeletal muscle. Our observation indicates that fulminate PRIS can occur in an adult patient with an inborn OXPHOS defect and corroborates the hypothesis that PRIS is caused by inhibition of the OXPHOS system.

Array comparative genomic hybridization in male infertility.

BACKGROUND: Male infertility caused by a maturation arrest of spermatogenesis is a condition with an abrupt stop in spermatogenesis, mostly at the level of primary spermatocytes. The etiology remains largely unknown. METHODS: We focused on patients with a complete arrest at the spermatocyte level (n = 9) and used array comparative genomic hybridization to screen for deletions or duplications that might be associated with maturation arrest. Interesting copy number variations (CNVs) were further examined by using quantitative PCR. Where appropriate, the expression pattern was analyzed in multiple human tissues including the testis. RESULTS: A total of 227 CNVs were detected in the patient group. After the elimination of CNVs that were also present in the control group or that were not likely to be involved in male infertility, the remaining 11 regions were investigated more in detail. We first determined the expression pattern of seven genes, for which expression had not been reported to be investigated in testicular tissue, after which one region could be eliminated. Next, all 10 promising candidate regions were analyzed by quantitative PCR in a control population. CONCLUSIONS: Eight deletions/duplications were absent in our control group, and therefore might be linked with the male infertility in our patients. One of these alterations, however, has been detected in a proven fertile father group. Further research is necessary to determine the relationship between the observed genomic alterations and maturation arrest of spermatogenesis. Furthermore, several of the above genes have not been studied at the functional level and consequently, more research is required to determine their role in spermatogenesis.

TUBA1A mutations: from isolated lissencephaly to familial polymicrogyria.

BACKGROUND: Mutations in the TUBA1A gene have been reported in patients with lissencephaly and perisylvian pachygyria. METHODS: Twenty-five patients with malformations of cortical development ranging from lissencephaly to polymicrogyria were screened for mutations in TUBA1A. RESULTS: Two novel heterozygous missense mutations in TUBA1A were identified: c.629A>G (p.Tyr210Cys) occurring de novo in a boy with lissencephaly, and c.13A>C (p.Ile5Leu) affecting 2 sisters with polymicrogyria whose mother presented somatic mosaicism for the mutation. CONCLUSIONS: Mutations in TUBA1A have been described in patients with lissencephaly and pachygyria. We report a mutation in TUBA1A as a cause of polymicrogyria. So far, all mutations in TUBA1A have occurred de novo, resulting in isolated cases. This article describes familial recurrence of TUBA1A mutations due to somatic mosaicism in a parent. These findings broaden the phenotypic spectrum associated with TUBA1A mutations and have implications for genetic counseling.

Mutation analysis of three genes in patients with maturation arrest of spermatogenesis and couples with recurrent miscarriages.

The primary aim of this study was to gain more insight into maturation arrest of spermatogenesis (MA) and its relationship with mutations in genes essential for meiosis. The study also investigated the possibility that mutations in human meiosis genes cause a milder phenotype and that, in such cases, meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution causing miscarriage. Among 40 patients with MA, five changes were observed that also predicted alterations at the amino acid level. However, since these changes were also present in men with normozoospermia in equal frequencies, it was assumed that these changes are single nucleotide polymorphisms. Among 46 patients with recurrent miscarriages, two additional changes were detected predicting an alteration at the amino acid level. One change was detected in controls. However, the second heterozygous change, detected in a conserved functional domain of the SYCP3 gene, was absent in >200 controls. These preliminary results stress the need to further investigate the relationship between abnormalities in meiosis genes and the formation of gametes with abnormal chromosomal constitution. More research is also necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling.

Krabbe leukodystrophy in a selected population with high rate of late onset forms: longer survival linked to c.121G>A (p.Gly41Ser) mutation.

Krabbe leukodystrophy (KD) is a neurodegenerative lysosomal disorder caused by mutations in the galactocerebrosidase (GALC) gene. Different clinical forms are described based on the age at onset. In reported series, the early infantile form (EIKD) accounts for more than 90% of the cases. The rarer late onset forms (LOKD) become manifest later than 6 months up to the adult age. We report clinical, imaging, mutational analysis and geographic data in a large cohort of individuals with Krabbe disease examined over a 30-year period. Retrospective analyses of disease onset and long-term follow-up were conducted in 26 KD patients. Molecular analysis was performed in 12 patients and their families. Nine cases had EIKD, and 17 LOKD, accounting for two thirds of our series. No correlation was found between enzymatic activity, onset age and disease progression. Despite common geographical origin, only in a few cases could parental consanguinity be proven. The p.Gly41Ser mutation was associated with longer survival. A wide spectrum of LOKD is found despite similar genotype. Although current knowledge about onset age, residual enzyme activity and molecular analysis still fail to allow the identification of patient candidates for treatment, this information is valuable for long-term outcome prediction and could lead to reconsideration of inclusion criteria for bone marrow transplant (BMT) or other future therapeutic approaches.

Mutational study in the PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase complex deficiency.

We screened for PDHA1 mutations in 40 patients with biochemically demonstrated PDHc deficiency or strong clinical suspicion, and found changes with probable pathological significance in 20. Five patients presented new mutations: p.A169V, c.932_938del, c.1143_1144 ins24, c.1146_1159dup and c.510-30G> A, this latter is a new undescribed cause of exon 6 skipping. Another four mutations have been found, and previously reported, in our patients: p.H113D, p.P172L, p.Y243del and p.Y369Q. Eleven patients presented seven known mutations: p.R127Q, p.I166I, p.A198T, p.R263G, p.R302C, p.R378C and c.1142_1145dup. The latter three were found in more than one unrelated patient: p.R302C was detected in a heterozygous girl and a mosaic male, p.R378C in two males and finally, c.1142_1145dup in three females; only one in 20 mothers was found to be a carrier (p.R263G). Apart from those 20 patients, the only alteration detected in one girl with clear PDHc and PDH-E1 deficiency was the silent change c.396A> C (p.R132R), and other eight PDHc deficient patients carry combinations of known infrequent polymorphisms that are overrepresented among our 20 unsolved patients. The importance of these changes on PDH activity is unclear. Investigations in the other PDHc genes are in course in order to elucidate the genetic defect in the unresolved patients.

Association of parathyroid adenoma and familial hypocalciuric hypercalcaemia in a teenager.

OBJECTIVE: Familial hypocalciuric hypercalcaemia (FHH) is clinically characterized by mild to moderate parathyroid hormone (PTH)-dependent hypercalcaemia, autosomal dominant pattern of inheritance, and normal to frankly reduced urinary calcium excretion in spite of a high serum calcium (clearance (Ca)/clearance (Cr)<0.01). FHH has a benign course and should be differentiated from primary hyperparathyroidism. It is usually caused by a heterozygous loss-of-function mutation in the calcium-sensing receptor gene (CASR). DESIGN: We report the case of a 16-year-old patient with hypercalcaemia and a mixed family history of parathyroid adenoma and mild hypercalcaemia. Serum calcium was 14 mg/dl with a serum iPTH of 253 pg/ml. RESULTS: A neck 99mTc-sesta MIBI tomoscintigraphy showed a definite hyperactivity in the left upper quadrant. A surgical four-gland exploration confirmed a single parathyroid adenoma. After surgical resection of a left superior parathyroid adenoma, the patient's hypercalcemia improved but did not normalize, returning to a level typical of FHH. An inactivating mutation in exon 4 of the CASR gene, predicting a p.Glu297Lys amino acid substitution was found. CONCLUSIONS: Thus, this 16-year old patient presented with the association of FHH and a single parathyroid adenoma. The young age of the patient and the association of parathyroid adenoma and FHH in his grandmother argue for a causal link between CASR mutation and parathyroid adenoma in this family. This case contributes to illustrate the expanding clinical spectrum of CASR loss-of-function mutations.

Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects.

BACKGROUND: Mitochondrial diseases display a heterogeneous spectrum of clinical phenotypes and therefore the identification of the underlying gene defect is often a difficult task. AIMS: To develop an immunohistochemical approach to stain skeletal muscle for the five multi-protein complexes that organise the oxidative phosphorylation (OXPHOS) in order to improve the diagnostic workup of mitochondrial defects. METHODS: OXPHOS complexes were visualised in skeletal muscle tissue using antibodies directed against different subunits. The staining patterns of patients with heteroplasmic defects in mtDNA tRNA genes were compared with those of normal and disease controls. RESULTS: Normal skeletal muscle displayed a checkerboard staining pattern for complexes I to V due to the higher mitochondrial content of slow muscle fibres versus fast fibres. In patients with tRNA defects, a much more heterogeneous staining pattern was observed for complex I (all six patients) and complex IV (4 of 6 patients): a mosaic staining pattern in which individual fibres displayed staining intensities that ranged from strong to negative. Ragged red fibres (RRFs) in patients with MERRF (myoclonic epilepsy and ragged red fibres) were all complex I and IV negative, while in patient with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) the majority of RRFs were complex I negative and complex IV positive. CONCLUSION: Immunohistochemical detection of OXPHOS complexes could represent a valuable additional diagnostic tool for the evaluation of mitochondrial cytopathy. The technique helps to detect heteroplasmic mtDNA defects. Staining for complex I in particular was able to identify two tRNA patients that stayed undetected with routine histochemical evaluation.

[Genetics and male infertility]. / Genetica van de mannelijke onvruchtbaarheid.

Infertility is a problem affecting many couples with a child wish. In about half of these couples a male factor is (co-) responsible for the fertility concern. For part of these patients a genetic factor will be the underlying cause of the problems. This paper gives an overview of the studies performed in the Department of Embryology and Genetics of the Vrije Universiteit Brussel and the Centre for Medical Genetics of UZ Brussel in order to gain more insight into the genetic causes of male infertility. The studies, focusing on men with fertility problems, can be subdivided into three groups: studies on deletions on the long arm of the Y chromosome, studies on X-linked genes and studies on autosomal genes. It is obvious that Yq microdeletions should be considered as a cause of male infertility. Only for patients with a complete AZFc deletion, a small number of spermatozoa can be retrieved. However, even for these patients assisted reproductive technologies are necessary. Complete AZF deletions are found in 4.6% of the patients visiting the centres for Reproductive Medicine and Medical Genetics of the UZ Brussel and for whom no other cause of the fertility problems have been detected. Taken into consideration this low prevalence of Yq microdeletions, it is obvious that also other factors, including genetic factors, must be causing fertility problems. Potentially, gr/gr deletions (partial deletions of the AZFc region) might influence the fertility status of the patients. It remains, however, unclear which of the genes located in the deleted regions are important for the progression of spermatogenesis, in case of partial or complete AZF deletions. In our studies we have also investigated mutations in genes located on the X chromosome. In analogy to the Y chromosome, the X chromosome is interesting in view of studying male infertility since men only have a single copy of the sex chromosomes. As a consequence, mutations in genes crucial for spermatogenesis will have an immediate impact on the sperm production. The genes NXF2, USP26 and TAF7L were investigated for the presence of mutations. All observed single nucleotide changes were also present in control samples, questioning their relationship with male infertility. We also studied five autosomal genes: SYCP3, MSH4, DNMT3L, STRA8 and ETV5. Only for the genes STRA8 and ETV5, changes were detected that were absent in a control population existing of men with normozoospermia. Functional analysis of the changes in ETV5 and the localization of the change observed in STRA8 showed that also these alterations were probably not the cause of the fertility problems in these men. It can be concluded that mutations are rarely detected in men with fertility problems. This low frequency of mutations has also been confirmed in several published studies. Therefore, further research is necessary to determine the impact of genetic causes on male infertility.

Is beta-glucuronidase a clinical useful biomarker for an acute organophosphorus poisoning?

beta-glucuronidase is considered a sensitive biomarker for acute organophosphorus poisoning. In this well-documented study, multiple plasma samples over time were collected. A decrease in plasma concentration of beta-glucuronidase was surprisingly observed, even within normal range. These findings do not support the hypothesis that beta-glucuronidase is a useful biomarker for acute organophosphorus poisoning in humans.

Preimplantation genetic diagnosis for cancer predisposition syndromes.

OBJECTIVES: Mutations in the APC, NF2 and BRCA1 genes cause adult-onset cancer predisposition syndromes. Prenatal diagnosis (PND) and selective pregnancy termination for adult-onset disorders is emotionally difficult and, in some cases, socially not well accepted. Preimplantation genetic diagnosis (PGD) appears as an attractive alternative to PND, as it ensures the establishment of a pregnancy free of the mutation from the onset, circumventing the potentially difficult decision of termination of pregnancy. METHODS: Development of single-cell PCRs using Epstein-Barr virus transformed lymphoblasts as single-cell model, followed by clinical application in PGD. RESULTS: A total of five duplex-PCRs were developed, three for adenomatous polyposis of the colon (APC), one for neurofibromatosis type 2 (NF2) and one for inherited breast and ovarian cancer caused by BRCA1 mutations. Eleven clinical cycles were performed, resulting in the birth of an unaffected girl. For one of the couples undergoing PGD for NF2, a spontaneous pregnancy ensued after five unsuccessful PGD cycles. The couple underwent chorionic villus sampling (CVS) and the application of the same protocol as used during PGD showed an unaffected fetus. CONCLUSION: In this work, we present the development and clinical application of PGD for three cancer predisposition syndromes.