Residual N-acetyl-α-glucosaminidase activity in fibroblasts correlates with disease severity in patients with mucopolysaccharidosis type IIIB.

BACKGROUND: Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare genetic disorder in which the deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU) results in the accumulation of heparan sulfate (HS), leading to progressive neurocognitive deterioration. In MPS IIIB a wide spectrum of disease severity is seen. Due to a large allelic heterogeneity, establishing genotype-phenotype correlations is difficult. However, reliable prediction of the natural course of the disease is needed, in particular for the assessment of the efficacy of potential therapies. METHODS: To identify markers that correlate with disease severity, all Dutch patients diagnosed with MPS IIIB were characterised as either rapid (RP; classical, severe phenotype) or slow progressors (SP; non-classical, less severe phenotype), based on clinical data. NAGLU activity and HS levels were measured in patients' fibroblasts after culturing at different temperatures. RESULTS: A small, though significant difference in NAGLU activity was measured between RP and SP patients after culturing at 37 °C (p < 0.01). Culturing at 30 °C resulted in more pronounced and significantly higher NAGLU activity levels in SP patients (p < 0.001) with a NAGLU activity of 0.58 nmol.mg-1.hr-1 calculated to be the optimal cut-off value to distinguish between the groups (sensitivity and specificity 100 %). A lower capacity of patients' fibroblasts to increase NAGLU activity at 30 °C could significantly predict for the loss of several disease specific functions. CONCLUSION: NAGLU activity in fibroblasts cultured at 30 °C can be used to discriminate between RP and SP MPS IIIB patients and the capacity of cells to increase NAGLU activity at lower temperatures correlates with disease symptoms.

Genotype phenotype correlations for hearing impairment: approaches to management.

Hearing impairment is an extremely heterogeneous disorder, with both environmental as well as genetic causes. This review describes the known genes involved in non-syndromic hearing impairment and their genotype-phenotype correlations where possible. Furthermore, some of the more frequent syndromic forms of hearing impairment are described, in particular where they overlap with the non-syndromic forms. Given the heterogeneity of the disorder, together with the indistinguishable phenotypes for many of the genes, it is suggested that testing for mutations is performed using massive parallel sequencing techniques, either by a large targeted set of genes or by an exome wide analysis.

Audiometric characteristics of a dutch family with a new mutation in GATA3 causing HDR syndrome.

We present the case of a Dutch family with a new mutation (c523_528dup) in GATA3 causing HDR syndrome. HDR syndrome is characterised by hypoparathyroidism, deafness and renal defects. In this study, we describe the audiometric characteristics of 5 patients from this family. Their hearing impairment was congenital, bilateral and symmetric. Audiograms showed mild-to-moderate hearing impairment with a flat audiogram configuration. Higher frequencies tended to be affected more strongly. Cross-sectional analyses showed no progression, and a mean audiogram was established. Psychophysical measurements in 3 HDR patients - including speech reception in noise, loudness scaling, gap detection and difference limen for frequency - were obtained to assess hearing function in greater detail. Overall, the results of the psychophysical measurements indicated characteristics of outer hair cell loss. CT scanning showed no anomalies in 3 of the HDR patients. Although 2 patients displayed vestibular symptoms, no anomalies in the vestibular system were found by vestibulo-ocular examination. Our results are in agreement with the theory that outer hair cell malfunctioning can play a major role in HDR syndrome.

CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype.

BACKGROUND: CHARGE syndrome is a highly variable, multiple congenital anomaly syndrome, of which the complete phenotypic spectrum was only revealed after identification of the causative gene in 2004. CHARGE is an acronym for ocular coloboma, congenital heart defects, choanal atresia, retardation of growth and development, genital hypoplasia, and ear anomalies associated with deafness. This typical combination of clinical features is caused by autosomal dominant mutations in the CHD7 gene. OBJECTIVE: To explore the emerging phenotypic spectrum of CHD7 mutations, with a special focus on the mild end of the spectrum. METHODS: We evaluated the clinical characteristics in our own cohort of 280 CHD7 positive patients and in previously reported patients with CHD7 mutations and compared these with previously reported patients with CHARGE syndrome but an unknown CHD7 status. We then further explored the mild end of the phenotypic spectrum of CHD7 mutations. RESULTS: We discuss that CHARGE syndrome is primarily a clinical diagnosis. In addition, we propose guidelines for CHD7 analysis and indicate when evaluation of the semicircular canals is helpful in the diagnostic process. Finally, we give updated recommendations for clinical surveillance of patients with a CHD7 mutation, based on our exploration of the phenotypic spectrum and on our experience in a multidisciplinary outpatient clinic for CHARGE syndrome. CONCLUSION: CHARGE syndrome is an extremely variable clinical syndrome. CHD7 analysis can be helpful in the diagnostic process, but the phenotype cannot be predicted from the genotype.

Hereditary leiomyomatosis and renal cell cancer in families referred for fumarate hydratase germline mutation analysis.

Heterozygous fumarate hydratase (FH) germline mutations cause hereditary leiomyomatosis and renal cell cancer (HLRCC), an autosomal dominant syndrome characterized by multiple cutaneous piloleiomyomas, uterine leiomyomas and papillary type 2 renal cancer. The main objective of our study was to evaluate clinical and genetic data from families suspected of HLRCC on a nationwide level. All families referred for FH mutation analysis in the Netherlands were assessed. We performed FH sequence analysis and multiplex ligation-dependent probe amplification. Families with similar FH mutations were examined for haplotype sharing. In 14 out of 33 families, we identified 11 different pathogenic FH germline mutations, including 4 novel mutations and 1 whole-gene deletion. Clinical data were available for 35 FH mutation carriers. Cutaneous leiomyomas were present in all FH mutation carriers older than 40 years of age. Eleven out of 21 female FH mutation carriers underwent surgical treatment for symptomatic uterine leiomyomas at an average of 35 years. Two FH mutation carriers had papillary type 2 renal cancer and Wilms' tumour, respectively. We evaluated the relevance of our findings for clinical practice and have proposed clinical diagnostic criteria, indications for FH mutation analysis and recommendations for management.

CHD7 mutations in patients initially diagnosed with Kallmann syndrome--the clinical overlap with CHARGE syndrome.

Kallmann syndrome (KS) is the combination of hypogonadotropic hypogonadism and anosmia or hyposmia, two features that are also frequently present in CHARGE syndrome. CHARGE syndrome is caused by mutations in the CHD7 gene. We performed analysis of CHD7 in 36 patients with KS and 20 patients with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) in whom mutations in KAL1, FGFR1, PROK2 and PROKR2 genes were excluded. Three of 56 KS/nIHH patients had de novo mutations in CHD7. In retrospect, these three CHD7-positive patients showed additional features that are seen in CHARGE syndrome. CHD7 mutations can be present in KS patients who have additional features that are part of the CHARGE syndrome phenotype. We did not find mutations in patients with isolated KS. These findings imply that patients diagnosed with hypogonadotropic hypogonadism and anosmia should be screened for clinical features consistent with CHARGE syndrome. If such features are present, particularly deafness, dysmorphic ears and/or hypoplasia or aplasia of the semicircular canals, CHD7 sequencing is recommended.

Mutations in CHD7 in patients with CHARGE syndrome cause T-B + natural killer cell + severe combined immune deficiency and may cause Omenn-like syndrome.

More than 11 genetic causes of severe combined immunodeficiency (SCID) have been identified, affecting development and/or function of T lymphocytes, and sometimes B lymphocytes and natural killer (NK) cells. Deletion of 22q11.2 is associated with immunodeficiency, although less than 1% of cases are associated with T-B + NK + SCID phenotype. Severe immunodeficiency with CHARGE syndrome has been noted only rarely Omenn syndrome is a rare autosomal recessive form of SCID with erythroderma, hepatosplenomegaly, lymphadenopathy and alopecia. Hypomorphic recombination activating genes 1 and 2 mutations were first described in patients with Omenn syndrome. More recently, defects in Artemis, RMRP, IL7Ralpha and common gamma chain genes have been described. We describe four patients with mutations in CHD7, who had clinical features of CHARGE syndrome and who had T-B + NK + SCID (two patients) or clinical features consistent with Omenn syndrome (two patients). Immunodeficiency in patients with DiGeorge syndrome is well recognized--CHARGE syndrome should now be added to the causes of T-B + NK + SCID, and mutations in the CHD7 gene may be associated with Omenn-like syndrome.

Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene.

BACKGROUND: CHARGE syndrome is a non-random clustering of congenital anomalies including coloboma, heart defects, choanal atresia, retarded growth and development, genital hypoplasia, ear anomalies, and deafness. A consistent feature in CHARGE syndrome is semicircular canal hypoplasia resulting in vestibular areflexia. Other commonly associated congenital anomalies are facial nerve palsy, cleft lip/palate, and tracheo-oesophageal fistula. Specific behavioural problems, including autistic-like behaviour, have been described. The CHD7 gene on chromosome 8q12.1 was recently discovered as a major gene involved in the aetiology of this syndrome. METHODS: The coding regions of CHD7 were screened for mutations in 107 index patients with clinical features suggestive of CHARGE syndrome. Clinical data of the mutation positive patients were sampled to study the phenotypic spectrum of mutations in the CHD7 gene. RESULTS: Mutations were identified in 69 patients. Here we describe the clinical features of 47 of these patients, including two sib pairs. Most mutations were unique and were scattered throughout the gene. All patients but one fulfilled the current diagnostic criteria for CHARGE syndrome. No genotype-phenotype correlations were apparent in this cohort, which is best demonstrated by the differences in clinical presentation in sib pairs with identical mutations. Somatic mosaicism was detected in the unaffected mother of a sib pair, supporting the existence of germline mosaicism. CONCLUSIONS: CHD7 mutations account for the majority of the cases with CHARGE syndrome, with a broad clinical variability and without an obvious genotype-phenotype correlation. In one case evidence for germline mosaicism was provided.

[From gene to disease: adrenogenital syndrome and the CYP21A2 gene]. / Van gen naar ziekte; het adrenogenitaal syndroom en het CYP21A2-gen.

Congenital adrenal hyperplasia (CAH) is a disorder of adrenal steroid synthesis. In more than 90% of cases CAH is caused by CYP21 (21-hydroxylase) deficiency leading to impaired cortisol and aldosterone synthesis and an increase in ACTH secretion. This then leads to stimulation of the adrenal gland and overproduction of androgens with virilisation of female external genitalia. The CYP21 enzyme consists of 495 amino acids and is encoded by the CYP21A2 gene located on chromosome 6p21.3 close to a 98% homologous pseudogene (CYP21p). The pseudogene contains several inactivating mutations that may be transferred to the active CYP21A2 gene by gene conversion (more than 60% of the affected alleles) or gene deletion (30% of the affected alleles). The severity of the disease depends on the degree of CYP21 deficiency. The diagnosis can be made by measuring levels of 17-hydroxyprogesterone and androstenedione in serum.

Erythropoiesis in myelodysplastic syndrome: expression of receptors for erythropoietin and kit ligand.

Ineffective erythropoiesis due to an impaired response to erythropoietin (EPO) is a prominent abnormality in myelodysplastic syndromes (MDS). The growth factor kit ligand (KL) may restore the in vitro erythroid colony-forming response to EPO in a subset of patients. The inability of MDS erythroid progenitors to react properly to EPO and/or KL has not been resolved. We have investigated erythropoietin receptor (EPO-R) and KL receptor (c-kit) expression in 15 cases of MDS by FACS analysis. The percentage of bone marrow cells expressing the EPO-R from patients with MDS were comparable to normal marrow. No apparent correlation was found between the number of MDS cells coexpressing the EPO-R and CD34 and impaired erythroid response. C-kit was expressed in most MDS patients, including those not responding to KL in EPO-induced cultures. In nine MDS cases the different splice variants of the EPO-R were analyzed. MDS cells, like normal marrow, expressed the full length EPO-R. These results show that impaired erythroid response in MDS cannot be explained by a quantitative lack of receptors for EPO or KL and that most likely suppression of erythroid response is caused by defective receptor signalling following ligand binding, representing a functional defect within the receptor itself or at a level downstream of the receptor.

Identical fusion transcript associated with different breakpoints in the AML1 gene in simple and variant t(8;21) acute myeloid leukemia.

Fluorescence in situ hybridization (FISH) and/or RNA-based polymerase chain reaction (RT-PCR) were used to analyze the breakpoints within the AML1 gene and the AML1 fusion transcripts in t(8;21) acute myeloid leukemia (AML). Twenty-two patients presented with the simple t(8;21)(q22;q22) and one with a complex variant t(8;2;16;21). In eight cases we used FISH with AML1 cosmid probes on metaphase chromosomes as well as RT-PCR to detect the junctions of MAL1/CDR (ETO,MTG8). Five cases were analyzed by FISH alone and ten cases by RT-PCR alone. By FISH we could identify three groups according to the distribution of the fluorescent signal. Signals were found in group 1 on chromosomes 21 and 21q+, in group 2 on chromosomes 21, 21q+ and 8q- and in group 3 on chromosomes 21 and 8q-. In all groups we could detect an identical AML1/CDR fusion transcript. This transcript showed splicing of AML1 exon 5 onto CDR. Thus regardless of the heterogeneity suggested by FISH, all the breakpoints in the AML1 gene were clustered in the same intro between exons 5 and 6. Our results bring to over one hundred the number of t(8;21) cases in which an identical translocation could be detected at molecular level by RT-PCR. The high sensitivity of the technique makes it suitable for the diagnosis of this translocation in different stages of the disease. The impact of the molecular detection of t(8;21) cells in clinical remission as far as the treatment and the management of the disease are concerned deserves further discussion.

Interleukin-7 signaling in human B cell precursor acute lymphoblastic leukemia cells and murine BAF3 cells involves activation of STAT1 and STAT5 mediated via the interleukin-7 receptor alpha chain.

Interleukin-7 (IL-7) stimulates the proliferation of normal and leukemic B and T cell precursors and T lymphocytes. Activation of the JAK/STAT pathway has been implicated in IL-7R signaling. We investigated which STAT complexes are formed upon stimulation of B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells with IL-7. Gel retardation assays with STAT-binding oligonucleotides showed that IL-7 induces the formation of two major STAT complexes in BCP-ALL cells. Supershifts with anti-STAT antibodies identified these as STAT1 and STAT5 complexes. This pattern of STAT activation was seen in all BCP-ALL cases that respond to IL-7 in proliferation assays. IL-7 also induced STAT/DNA binding in BCP-ALL cases that failed to proliferate in response to IL-7, suggesting that the ability of IL-7R to activate the JAK/STAT pathway per se is not sufficient for proliferation induction. To determine the contribution of the cytoplasmic domain of the IL-7 receptor alpha chain (IL-7R alpha) to activation of STAT proteins, transfectants of the murine pro-B cell line BAF3 were made that express chimeric receptors consisting of the extracellular domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) and the transmembrane and intracellular domains of human IL-7R alpha. Activation of the chimeric G-CSF-R/IL-7R alpha with G-CSF resulted in a full proliferative response and induced the phosphorylation of JAK1 but not JAK2. Major STAT complexes activated by G-CSF-R/IL-7R alpha contained STAT1 or STAT5, while some formation of STAT3-containing complexes was also seen. These findings establish that STAT1 and STAT5, and possibly STAT3, are activated upon stimulation of precursor B cells with IL-7. The data further indicate that the IL-7R alpha chains are directly involved in the activation of JAKs and STATs and have a major role in proliferative signaling in precursor B cells.

Teunissen-Cremers syndrome: a clinical, surgical, and genetic report.

OBJECTIVE: To describe clinical and radiologic features, results of ear surgery, and genetic analysis in three families with Teunissen-Cremers syndrome. DESIGN: Case series. SETTING: Tertiary referral center. BACKGROUND: The NOG gene encodes the protein noggin, which has antagonist action in osteogenesis. Malformation of bones and joints may result from defects in noggin. Teunissen-Cremers syndrome is caused by mutations in the NOG gene. Two mutations in this gene were reported previously. The proximal symphalangism-hearing impairment syndrome, also caused by mutations in the NOG gene, is characterized by proximal symphalangism, conductive hearing loss, and occasionally synostoses. METHODS: We examined nine affected members of three Dutch families. Reconstructive middle ear surgery was performed in five patients (nine ears), and we sequenced the NOG gene in these families. RESULTS: Affected members had conductive hearing impairment, hyperopia, and broad thumbs and first toes with brachytelephalangia. Surgery manifested stapes ankylosis with additional incudal fixation frequently in the fossa incudis. Air-bone gaps decreased to less than 10 dB in six ears. Genetic analysis revealed three new mutations in the NOG gene. CONCLUSION: The Teunissen-Cremers syndrome is an entity in its clinical presentation, distinct from other syndromes with proximal symphalangism and hearing impairment. So far, in five families with Teunissen-Cremers syndrome, four truncating mutations and one amino acid substitution were found in the NOG gene. The majority of other mutations found in this gene are missense mutations, which might result in some residual protein activity. Reconstructive middle ear surgery is an option for treatment.

[From gene to disease; mutations in the SLC12A3 gene as the cause of Gitelman's syndrome]. / Van gen naar ziekte; mutaties in het SLC12A3-gen als oorzaak van het syndroom van Gitelman.

Gitelman's syndrome is characterised by persistent hypokalaemia, hypomagnesaemia and hypocalciuria (OMIM 263800). This rare autosomal recessive disorder is caused by renal Na+, Cl-, K+ and Mg2+ wasting. Other typical features include hypocalciuria and an intact renal concentrating ability. Gitelman's syndrome is caused by mutations in the SLC12A3 gene, encoding the thiazide-sensitive sodium-chloride co-transporter (NCC). NCC is located in the distal convoluted tubule of the kidney, a segment known to play an important role in active magnesium reabsorption in the nephron. The exact mechanisms underlying hypomagnesaemia and hypocalciuria in Gitelman's syndrome are still poorly understood, but point to enhanced proximal Na+ and Ca2+ reabsorption and apoptosis of distal convoluted tubule cells.

The C-terminal cytoplasmic region of the granulocyte colony-stimulating factor receptor mediates apoptosis in maturation-incompetent murine myeloid cells.

Granulocyte colony-stimulating factor (G-CSF) promotes the survival and proliferation of myeloid progenitors and induces maturation of these cells toward terminally differentiated neutrophils. Using transfectants of the murine IL-3-dependent myeloid cell line 32D that express the human G-CSF receptor (32D/WT cells), we show here that G-CSF can also exert adverse effects on myeloid cell survival. Although initially enhancing IL-3-driven proliferation of 32D/WT cells, G-CSF strongly inhibited cell survival at later stages of culture. The loss of viability of 32D/WT cells following sustained G-CSF stimulation was not accompanied by progressive neutrophilic maturation. Instead, 32D/WT cells exhibited features characteristic of apoptosis. The apoptosis-inducing effect of G-CSF was seen at concentrations of IL-3 that could support long-term proliferation and survival of 32D/WT cells in the absence of G-CSF. Experiments with 32D cells expressing mutant forms of the G-CSF receptor revealed that the death signals were mediated exclusively through the membrane-distal cytoplasmic part of the G-CSF receptor, a region also involved in maturation signaling.

Novel PTEN mutations in patients with Cowden disease: absence of clear genotype-phenotype correlations.

Cowden disease (CD) is characterised by multiple hamartomas in a variety of tissues. The pathological hallmark is the presence of a number of trichilemmomas. Several neurological symptoms are also part of CD with megalencephaly and Lhermitte-Duclos disease (LDD) as the most important features. Early recognition of CD patients is important because of the increased risk of developing malignancies. Breast cancer is the most frequent malignancy, but also urogenital, digestive tract, and thyroid cancers are found with higher frequencies. CD was localised to chromosome 10q23 and the PTEN gene (also known as MMAC1 or TEP1) was shown to be involved. Germline mutations were identified in both familial and sporadic CD patients. We identified eight PTEN mutations, of which seven were novel, in 13 CD patients. Combined with previous data we have identified 17 independent CD mutations. Gross DNA alterations in CD patients were not detected. Genotype-phenotype relations are discussed. The only correlation suggested to exist is that missense mutations are not detected in LDD patients. However, larger numbers are needed to confirm this. Association of PTEN mutations and the occurrence of malignant breast disease found in an earlier study cannot be confirmed. Clinical features of five CD patients without a PTEN mutation in the coding sequence do not differ from CD patients with a PTEN mutation. Furthermore, it is likely that we have identified the majority of CD patients in the Netherlands. From this we estimate that CD has a prevalence of about 1 in 250,000 in the Dutch population with a low mutation frequency.

PTEN mutation in a family with Cowden syndrome and autism.

We report on a mother and son with Cowden syndrome and a PTEN mutation. The boy also exhibits autistic behavior and mental retardation, while his mother has a normal intelligence and social interaction pattern. We review the scanty literature data on the association of Cowden syndrome and autism and emphasize that the association of progressive macrocephaly and pervasive developmental disorder seems to be an indication for screening for PTEN mutations.

[From gene to disease; deletion of the DAZ-gene from the Y-chromosome in oligo- or azoospermia ]. / Van gen naar ziekte; deletie van het DAZ-gen op het Y-chromosoom en oligo- of azoöspermie.

DAZ gene deletions at the azoospermia factor (AZF) locus on the Y chromosome, have been implicated as one of the major causes of idiopathic male subfertility. Deletions of the entire DAZ gene have been reported in azoospermia as well as in oligozoospermia. The DAZ gene encodes a RNA binding protein which is expressed exclusively in germ cells. The exact biological role and function of the DAZ protein has yet to be resolved.