Summary DAX1 (NR0B1) is an orphan nuclear receptor, which plays an important role in development and function of the adrenal glands and gonads. Mutations in DAX1 cause X-linked adrenal hypoplasia congenita (X-linked AHC), which is characterized by adrenal insufficiency (AI) and hypogonadotropic hypogonadism (HHG). Affected boys present with adrenal failure usually in childhood and, later in life, with delayed puberty. However, patients with a late-onset form of X-linked AHC have also been described in the past years. We report a male patient who presented with symptoms of an adrenal crisis at the age of 38 years and was later diagnosed with HHG. Family history was positive with several male relatives diagnosed with AI and compatible with the assumed X-chromosomal inheritance of the trait. Direct sequencing of DAX1 of the patient revealed a hemizygous cytosine-to-thymine substitution at nucleotide 64 in exon 1, which creates a novel nonsense mutation (p.(Gln22*)). In order to compare the clinical presentation of the patient to that of other patients with X-linked AHC, we searched the electronic database MEDLINE (PubMed) and found reports of nine other cases with delayed onset of X-linked AHC. In certain cases, genotype–phenotype correlation could be assumed. Learning points: X-linked AHC is a rare disease characterized by primary AI and hypogonadotropic hypogonadism (HHG). The full-blown clinical picture is seen usually only in males with a typical onset in childhood. Patients with a late-onset form of X-linked AHC have also been described recently. Being aware of this late-onset form might help to reach an early diagnosis and prevent life-threatening adrenal crises. Adult men with primary AI of unknown etiology should be investigated for HHG. Detecting a DAX1 mutation may confirm the clinical diagnosis of late-onset X-linked AHC. In relatives of patients with genetically confirmed X-linked AHC, targeted mutation analysis may help to identify family members at risk and asymptomatic carriers, and discuss conscious family planning.

Fabry disease (FD) is a lysosomal storage disorder, which develops due to a deficiency in the hydrolytic enzyme, α-galactosidase A (α-Gal A). Alpha-Gal A hydrolyzes glycosphingolipid globotriaosylceramide (Gb3), and an α-Gal A deficiency leads to Gb3 accumulation in tissues and cells in the body. This pathology is likely to involve multiple systems, but it is generally considered to affect primarily vascular endothelium. In this study, we investigated mutations in the GLA gene, which encodes α-Gal A, in Mexican families with FD. We included seven probands with FD that carried known mutations. We analysed pedigrees of the probands, and performed molecular screening in 65 relatives with the potential of carrying a GLA mutation. Five mutations (P40S, IVS4 +4, G328V, R363H, R404del) were detected in seven unrelated Mexican families with the classic FD phenotype. Of the 65 relatives examined, 42 (64.6%) had a GLA gene mutation. In summary, among seven Mexican probands with FD, 65 relatives were at risk of carrying a known GLA mutation, and molecular screening identified 42 individuals with the mutation. Thus, our findings showed that it is important to perform molecular analysis in families with FD to detect mutations and to provide accurate diagnoses for individuals that could be affected.

BACKGROUND:Early-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family. METHODS:A panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation. RESULTS:Onset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141_142delTG (p.Arg50Alafs*103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice. CONCLUSION:The function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness.

Background: Glioblastomas (GBM) are often characterized by an elevated expression of the epidermal growth factor receptor variant III (EGFRvIII). We used GBM cell lines with native EGFRvIII expression to determine whether this EGFR variant affects radiosensitivity with or without EGFR targeting.Methods: Experiments were performed with GBM cell lines lacking (LN229, U87MG, U251, CAS-1) or endogenously expressing EGFRvIII (BS153, DKMG). The two latter cell lines were also used to establish sublines with a low (-) or a high proportion (+) of cells expressing EGFRvIII. EGFR signaling and the cell cycle were analyzed using Western blot and flow cytometry; cell survival was assessed by colony forming assay and double-strand break repair capacity by immunofluorescence.Results: DKMG and BS153 parental cells with heterogeneous EGFRvIII expression were clearly more radiosensitive compared to other GBM cell lines without EGFRvIII expression. However, no significant difference was observed in cell proliferation, clonogenicity or radiosensitivity between the EGFRvIII- and + sublines derived from DKMG and BS153 parental cells. Expression of EGFRvIII was associated with decreased DSB repair capacity for BS153 but not for DKMG cells. The effects of EGFR targeting by gefitinib alone or in combination with irradiation were also found not to depend on EGFRvIII expression. Gefitinib was only observed to influence the proliferation of EGFRvIII-BS153 cells.Conclusion: The data indicate that EGFRvIII does not alter radiosensitivity with or without anti-EGFR treatment.

Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase (IDS). The human IDS gene is located in chromosome Xq28. This is the first report of genotype and phenotype characterization of 49 Hunter patients from 40 families of Argentina. Thirty different alleles have been identified, and 57% were novel. The frequency of de novo mutations was 10%. Overall, the percentage of private mutations in our series was 75%.

Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.

Background and purposeThere is a great need to improve the outcome of locoregionally advanced squamous cell carcinomas of the head and neck (HNSCC). Standard treatment includes a combination of surgery, radio- and chemotherapy. The addition of molecular targeting agents to conventional treatment may improve outcomes. In this study the Raf inhibitor sorafenib was used to increase the radiosensitivity of HNSCC cell lines.Material and methodsIn a panel of six cell lines (A549, FaDu, UTSCC 60A, UTSCC 42A, UTSCC 42B, UTSCC 29) radiosensitivity was measured by colony formation assay and apoptosis and cell cycle analysis were performed by flow cytometry. DNA repair was analyzed by 53BP1 immunohistochemistry.ResultsSorafenib added prior to irradiation resulted in an increased cellular radiosensitivity (DEF0.5=1.11–1.84). Radiosensitization was not caused by an enhanced rate of apoptosis or cell cycle effects. In contrast, sorafenib was shown for the first time to block the repair of DNA double-strand breaks (DSB).ConclusionOur data suggest that sorafenib may be used to overcome the radioresistance of HNSCC through the inhibition of DSB repair.

We report a Mexican girl showing the full blown clinical picture of mucopolysaccharidosis type II (MPSII). Iduronate-2-sulfatase (IDS) activity was low and she carried a heterozygous de novo c.1327C>T transition in exon 9, that changes codon 443 for a premature stop (TGA; p.Arg443∗). Analysis of X-chromosome inactivation in androgen receptor (AR) locus showed a highly skewed ratio of 92:8 suggesting a functional hemizygosity with dominant expression of the mutant IDS and explaining the disease manifestation. This is one of the rare cases of females affected by MPSII due to the combined effect of a skewed X-chromosome inactivation and a de novo IDS mutation. We recommend that clinicians should consider the diagnosis of MPSII even in a girl without positive family history for this condition.

Retinitis pigmentosa (RP) is a group of human retinal disorders, with more than 100 genes involved in retinal degeneration. Canine and murine models are useful for investigating human RP based on known, naturally occurring mutations. In Schapendoes dogs, for example, a mutation in the CCDC66 gene has been shown to cause autosomal recessively inherited, generalized progressive retinal atrophy (gPRA), the canine counterpart to RP. Here, a novel mouse model with a disrupted Ccdc66 gene was investigated to reveal the function of protein CCDC66 and the pathogenesis of this form of gPRA. Homozygous Ccdc66 mutant mice lack retinal Ccdc66 RNA and protein expression. Light and electron microscopy reveal an initial degeneration of photoreceptors already at 13 days of age, followed by a slow, progressive retinal degeneration over months. Retinal dysfunction causes reduced scotopic a-wave amplitudes, declining from 1 to 7 months of age as well as an early reduction of the photopic b-wave at 1 month, improving slightly at 7 months, as evidenced by electroretinography. In the retina of the wild-type (WT) mouse, protein CCDC66 is present at highest levels after birth, followed by a decline until adulthood, suggesting a crucial role in early development. Protein CCDC66 is expressed predominantly in the developing rod outer segments as confirmed by subcellular analyses. These findings illustrate that the lack of protein CCDC66 causes early, slow progressive rod-cone dysplasia in the novel Ccdc66 mutant mouse model, thus providing a sound foundation for the development of therapeutic strategies.

Practically 100% in males and females if both sequencing and dosage testing are performed. Practically 100%. Index case in that family had been tested: Practically 100% in males and females if both sequencing and dosage testing are performed. Index case in that family had not been tested: Practically 100% in males and females if both sequencing and dosage testing are performed. Yes, adequate therapeutic options (see 3.1.4), informed family planning. If the test result is negative (please describe): ‘Relief’ with regard to the familial risk, informed family planning. Please assume that the result of a genetic test has no immediate medical consequences. Is there any evidence that a genetic test is nevertheless useful for the patient or his/her relatives? (Please describe) Results of molecular genetic diagnostics may influence family planning. Often it helps clarifying the situation and allows patients and relatives to choose the most appropriate options. The authors declare no conflict of interest. This work was supported by EuroGentest, an EU-FP6 supported NoE, contract number 512148 (EuroGentest Unit 3: ‘Clinical genetics, community genetics and public health’, Workpackage 3.2).

History and admission finding: A 48-year-old patient (case 1) with progressive stiffness and weakness of the legs and urge incontinence and a 38-year-old patient (case 2) with primary adrenal insufficiency and familial occurence adrenoleukodystorphy (ALD) presented for further evaluation.Investigations: Laboratory values in case 1 revealed primary adrenal insufficiency (decreased cortisol and increased corticotropin) and increase of the very long chain fatty acids (VLCFA). Clinical examination revealed paraspasticity and neurography showed demyelinating motor neuropathy of the lower extremities. Case 2 also showed pyramidal tract affection to the legs, reduced nerve conduction velocity of the tibial and sural nerves and increased VLCFA.Diagnosis, treatment and course: These findings were consistent with the diagnosis of the adult form of ALD. In case 1, supplementation therapy with hydrocortisone and fludrocortisone was initiated, resulting in marked alleviation of the clinical symptoms. In contrast, urge incontinence did not significantly improve despite multimodal drug treatment with Lorenzo's oil and restriction of dietary VLCFA were initiated and supplementation treatment for adrenal insufficiency was continued. On a follow-up visit one year later, the patients' conditions were stable.Conclusions: ALD comprises a wide spectrum of clinical symptoms. Adrenal insufficiency may precede neurological symptoms (main neurological symptoms: paraspasticity and/or demyelinating neuropathy). Regular testing for adrenal insufficiency and adequate supplementation therapy are mandatory. In most patients suffering from ALD therapeutic options comprise restriction of dietary VLCFA, but their clinical efficacy remains to be proven. Haematopoetic stem cell transplantation is only recommended in the initial state of cerebral involvement.

Anamnese und klinischer Befund: Ein 48-jahriger Patient (Fall 1) mit zunehmender Steifigkeit und Schwache in den Beinen und Urge-Inkontinenz sowie ein 38-jahriger Patient (Fall 2) mit primarer Nebennierenrindeninsuffizienz und familiar gehauftem Auftreten einer Adrenoleukodystrophie (ALD) stellten sich zur weiteren Abklarung vor. Untersuchungen: Die Laborwerte bei Fall 1 ergaben eine primare Nebenniereninsuffizienz (vermindertes Kortisol und erhohtes Kortikotropin) sowie eine Erhohung der uberlangkettigen Fettsauren. Klinisch zeigte sich eine Paraspastik und neurographisch eine demyelinisierende motorische Neuropathie an den Beinen. Bei Fall 2 bestanden ebenfalls eine Pyramidenbahnlasion zu den Beinen, neurographisch verlangsamte Leitungsgeschwindigkeiten des N. tibialis und N. suralis und eine Erhohung der uberlangkettigen Fettsauren. Diagnose, Therapie und Verlauf: Die Untersuchungsergebnisse waren mit der Diagnose einer ALD vom Erwachsenentyp vereinbar. Bei Fall 1 wurde eine Substitutionstherapie mit Hydrokortison und Fludrokortison begonnen, darunter kam es zu einer deutlichen Verbesserung der Symptomatik. Hingegen konnte die Urgeinkontinenz trotz einer multimodalen Medikation nicht gunstig beeinflusst werden. Bei Fall 2 wurde unter Fortfuhrung der Glukokortikoid- und Mineralokortikoidsubstitutionstherapie die Anwendung von Lorenzos Ol und eine Nahrungsrestriktion fur uberlangkettige Fettsauren initiiert. Eine Untersuchung ein Jahr spater ergab einen stabilen Befund. Folgerung: Die ALD umfasst ein weites Spektrum an Symptomen. Eine Nebennierenrindeninsuffizienz kann sich vor dem Auftreten neurologischer Symptome (hier fuhrend: Paraspastik und/oder demyelinsierende Neuropathie) manifestieren. Wichtig ist das regelmasige Screening auf eine Nebennierenrindeninsuffizienz und eine ausreichende Substitutionstherapie. Therapeutisch ist eine Nahrungsrestriktion fur uberlangkettige gesattigte Fettsauren zu erwagen, aussagekraftige Studien zur Wirksamkeit stehen jedoch bislang aus. Eine hamatopoetische Stammzelltransplantation ist nur im Fruhstadium einer zerebralen Beteiligung zu diskutieren. History and admission findings: A 48-year-od patient (case 1) with progressive stiffness and weakness of the legs and urge incontinence and a 38-year-old patient (case 2) with primary adrenal insufficiency and familial occurrence of adrenoleukodystrophy (ALD) presented for further evaluation. Investigations: Laboratory values in case 1 revealed primary adrenal insufficiency (decreased cortisol and increased corticotropin) and increase of the very long chain fatty acids (VLCFA). Clinical examination revealed paraspasticity and neurography showed demyelinating motor neuropathy of the lower extremities. Case 2 also showed pyramidal tract affection to the legs, reduced nerve conduction velocity of the tibial and sural nerves and increased VLCFA. Diagnosis, treatment and course: These findings were consistent with the diagnosis of the adult form of ALD. In case 1, supplementation therapy with hydrocortisone and fludrocortisone was initiated, resulting in marked alleviation of the clinical symptoms. In contrast, urge incontinence did not significantly improve despite multimodal drug treatment. In case 2, treatment with Lorenzo‘s oil and restriction of dietary VLCFA were initiated and supplementation treatment for adrenal insufficiency was continued. On a follow-up visit one year later, the patients‘ conditions were stable. Conclusions: ALD comprises a wide spectrum of clinical symptoms. Adrenal insufficiency may precede neurological symptoms (main neurological symptoms: paraspasticity and/or demyelinating neuropathy). Regular testing for adrenal insufficiency and adequate supplementation therapy are mandatory. In most patients suffering from ALD therapeutic options comprise restriction of dietary VLCFA, but their clinical efficacy remains to be proven. Haematopoetic stem cell transplantation is only recommended in the initial stage of cerebral involvement.

Ein Mangel des lysosomalen Enzyms Alpha-Galactosidase A führt zur Erkrankung Morbus Fabry (OMIM 301500), die charakterisiert ist durch Akkumulation von Glykoshingolipiden (Gb3) in verschiedensten Zelltypen. Obwohl die Pathologie der X-chromosomal vererbten Erkrankung bereits beim Feten beginnt, ist der Beginn der Erkrankung mit ersten Symptomen erst Jahre später im Alter von 6,7 (±3.4) Jahren bei Jungen und 7.8 (±4.5) Jahren bei Mädchen beschrieben. Frühe Manifestationen der Erkrankung bei Kindern beinhalten akute und chronische neuropathische Schmerzen (Akroparästhesien=typische brennende Schmerzen an Handflächen und Fußsohlen), Hypohidrose, Angiokeratome und gastrointestinale Symptome. Weitere typische Symptome sind: Cornea verticillata, Angiokeratome und Tinnitus. Als Spätkomplikationen auf Grund der Akkumulation von Gb3 kann es zu Kardiomyopathie, Apoplex sowie Niereninsuffizienz bis hin zum Nierenversagen kommen. Hier berichten wir über die Tochter einer symptomatischen Patientin mit Morbus Fabry, die schon im Alter von 2 Jahren über Schmerzen an den Füßen geklagt hat und die Familiäre Mutation p.N329I des GLA-Genes trägt. Das Kind zog immer Schuhe und Strümpfe aus und verweigerte das Laufen. Im Sommer fiel eine Gesichtsröte auf (Symptom der Hypohidrose). Die brennenden Schmerzen wurden im Verlauf schlimmer so dass die Behandlung mit NSAIDs erforderlich war. Akroparästhesien werden im Kindesalter oft übersehen. Die körperliche Untersuchung kann nicht immer Hinweise auf die korrekte Diagnose geben. EMG und NLG zeigen keine Veränderungen, da die Fabry Erkrankung primär die small fibres betrifft. In Deutschland ist die Enzymersatztherapie erst ab dem Alter von 7 Jahren zugelassen. Wir empfehlen, Kinder mit Morbus Fabry regelmäßig in einem Zentrum für lysosomale Stoffwechselerkrankungen vorzustellen. Ein früherer Therapiebeginn soll diskutiert werden, um evtl. lebenslangen Akroparästhesien oder anderen systemische Komplikationen vorzubeugen.