Evaluation of impact of anti-idursulfase antibodies during long-term idursulfase enzyme replacement therapy in mucopolysaccharidosis II patients.

OBJECTIVES: This 109-week, nonrandomized, observational study of mucopolysaccharidosis II (MPS II) patients already enrolled in the Hunter Outcome Survey (HOS) (NCT00882921), assessed the long-term immunogenicity of idursulfase, and examined the effect of idursulfase-specific antibody generation on treatment safety (via infusion-related adverse events [IRAEs]) and pharmacodynamics (via urinary glycosaminoglycans [uGAGs]). METHODS: Male patients ≥ 5 years, enrolled in HOS regardless of idursulfase treatment status were eligible. Blood/urine samples for anti-idursulfase antibody testing and uGAG measurement were collected every 12 weeks. RESULTS: Due to difficulties in enrolling treatment-naïve patients, data collection was limited to 26 enrolled patients of 100 planned patients (aged 5.1-35.5 years) all of whom were non-naïve to treatment. Fifteen (58%) patients completed the study. There were 11/26 (42%) seropositive patients at baseline (Ab +), and 2/26 (8%) others developed intermittent seropositivity by Week 13. A total of 9/26 patients (35%) had ≥ 1 sample positive for neutralizing antibodies. Baseline uGAG levels were low due to prior idursulfase treatment and did not change appreciably thereafter. Ab + patients had persistently higher uGAG levels at entry and throughout the study than Ab - patients. Nine of 26 (34%) patients reported IRAEs. Ab + patients appeared to have a higher risk of developing IRAEs than Ab - patients. However, the relative risk was not statistically significant and decreased after adjustment for age. CONCLUSIONS: 50% of study patients developed idursulfase antibodies. Notably Ab + patients had persistently higher average uGAG levels. A clear association between IRAEs and antibodies was not established.

The Long and Short of Genetic Counseling Summary Letters: A Case-control Study.

Genetic counseling summary letters are intended to reinforce information received during genetic counseling, but little information is available on patient/family responses to these letters. We conducted a case-control study to assess the effectiveness of two different letter formats. Parents of children receiving a new diagnosis were enrolled. The control group (n = 85) received a genetic counseling summary letter in a narrative format, 4-5 pages in length. After the control enrollment period, genetic counselors were trained by a professional medical writer to develop a concise letter format. The case group (n = 64) received a concise letter, approximately 1.5 pages in length, utilizing simple sentences, lay terms, and lists/bullet points. Parents completed a survey 4 weeks after the visit to rate the letter's format, usefulness, and their emotional reaction. Results show that parents in the case group rated the letter more highly (p = 0.023), particularly in the emotional response dimension (rating changes in anxiety, depression, fear, ability to cope, and confidence in response to the letter). Parents in the case group also rated the genetic counseling session more highly (p = 0.039). In the control group, parents without a college degree were more likely to rate the letter as too long and the level of medical detail as too high. In the case group, no significant differences were seen between parents with or without a college degree. These data suggest that a short genetic counseling summary letter is rated higher by parents, and is particularly associated with a more positive emotional reaction. A short letter format highlighting the basic facts related to the genetic condition may be more useful to parents of diverse educational backgrounds, and may support a positive emotional adaptation at the time of a new diagnosis. Genetic counselors may benefit from specific instruction in medical and educational writing.

Expert recommendations for the laboratory diagnosis of MPS VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This enzyme is required for the degradation of dermatan sulfate. In its absence, dermatan sulfate accumulates in cells and is excreted in large quantities in urine. Specific therapeutic intervention is available; however, accurate and timely diagnosis is crucial for maximal benefit. To better understand the current practices for diagnosis and to establish diagnostic guidelines, an international MPS VI laboratory diagnostics scientific summit was held in February of 2011 in Miami, Florida. The various steps in the diagnosis of MPS VI were discussed including urinary glycosaminoglycan (uGAG) analysis, enzyme activity analysis, and molecular analysis. The following conclusions were reached. Dilute urine samples pose a significant problem for uGAG analysis and MPS VI patients can be missed by quantitative uGAG testing alone as dermatan sulfate may not always be excreted in large quantities. Enzyme activity analysis is universally acknowledged as a key component of diagnosis; however, several caveats must be considered and the appropriate use of reference enzymes is essential. Molecular analysis supports enzyme activity test results and is essential for carrier testing, subsequent genetic counseling, and prenatal testing. Overall the expert panel recommends caution in the use of uGAG screening alone to rule out or confirm the diagnosis of MPS VI and acknowledges enzyme activity analysis as a critical component of diagnosis. Measurement of another sulfatase enzyme to exclude multiple sulfatase deficiency was recommended prior to the initiation of therapy. When feasible, the use of molecular testing as part of the diagnosis is encouraged. A diagnostic algorithm for MPS VI is provided.

A synonymous mutation in TCOF1 causes Treacher Collins syndrome due to mis-splicing of a constitutive exon.

Interpretation of the pathogenicity of sequence alterations in disease-associated genes is challenging. This is especially true for novel alterations that lack obvious functional consequences. We report here on a patient with Treacher Collins syndrome (TCS) found to carry a previously reported mutation, c.122C > T, which predicts p.A41V, and a novel synonymous mutation, c.3612A > C. Pedigree analysis showed that the c.122C > T mutation segregated with normal phenotypes in multiple family members while the c.3612A > C was de novo in the patient. Analysis of TCOF1 RNA in lymphocytes showed a transcript missing exon 22. These results show that TCS in the patient is due to haploinsufficiency of TCOF1 caused by the synonymous de novo c.3612A > C mutation. This study highlights the importance of clinical and pedigree evaluation in the interpretation of known and novel sequence alterations.

Genotype differences in cognitive functioning in Noonan syndrome.

Noonan syndrome (NS) is an autosomal-dominant genetic disorder associated with highly variable features, including heart disease, short stature, minor facial anomalies and learning disabilities. Recent gene discoveries have laid the groundwork for exploring whether variability in the NS phenotype is related to differences at the genetic level. In this study, we examine the influence of both genotype and nongenotypic factors on cognitive functioning. Data are presented from 65 individuals with NS (ages 4-18) who were evaluated using standardized measures of intellectual functioning. The cohort included 33 individuals with PTPN11 mutations, 6 individuals with SOS1 mutations, 1 individual with a BRAF mutation and 25 participants with negative, incomplete or no genetic testing. Results indicate that genotype differences may account for some of the variation in cognitive ability in NS. Whereas cognitive impairments were common among individuals with PTPN11 mutations and those with unknown mutations, all of the individuals with SOS1 mutations exhibited verbal and nonverbal cognitive skills in the average range or higher. Participants with N308D and N308S mutations in PTPN11 also showed no (or mild) cognitive delays. Additional influences such as hearing loss, motor dexterity and parental education levels accounted for significant variability in cognitive outcomes. Severity of cardiac disease was not related to cognitive functioning. Our results suggest that some NS-causing mutations have a more marked impact on cognitive skills than others.

Unique case of trisomy 2p24.3-pter with no associated monosomy.

We report a 3-year-old girl with trisomy 2p24.3-pter who presented with marked psychomotor delay and dysmorphic features. This patient represents the only known case of trisomy 2p24.3-2pter that does not involve an associated functional monosomy. In contrast to recent reports highlighting the fatal or serious complications in patients described as having partial trisomy 2p, this patient does not have significant birth defects or life threatening medical problems. Notably, this patient does not have a neural tube defect (NTD), which has been attributed to a putative locus at 2p24 in other patients with a partial trisomy 2p.

Regulation of the human long chain acyl-CoA dehydrogenase gene by nuclear hormone receptor transcription factors.

Mitochondrial fatty acid oxidation provides most of the energy required for myocardial function after birth. Long chain acyl-CoA dehydrogenase (LCAD) catalyzes the first step in the beta-oxidation spiral. Our objective was to define regulatory elements of the human LCAD gene required for high levels of expression in mature heart and to locate elements suppressing gene expression in the fetus. We characterized the human LCAD gene structure and used in vitro transfection into cardiomyocytes and hepatoma cells of LCAD genomic fragments fused to a reporter gene to examine the effects of putative regulatory elements on transcription. Binding of transcription factors to nuclear hormone receptor consensus DNA binding domains was studied by gel shift experiments. The 200 bp of the human LCAD gene immediately upstream of the transcription initiation site are sufficient to act as a minimal promoter for the gene and provide some tissue-specific positive regulatory elements. The region from -1800 bp to -250 bp contains elements which markedly suppress transcription, including nuclear hormone receptor response elements. The dominant interaction is with the repressor factor, chicken ovalbumin upstream promoter transcription factor. We conclude that the developmental and tissue-specific regulation of the human LCAD gene is mediated, in part, by these nuclear hormone receptor transcription factors.

Monosomy 9p24-->pter and trisomy 5q31-->qter: case report and review of two cases.

Partial deletion of the short arm of chromosome 9 (p24-->pter) and partial duplication of the long arm of chromosome 5 (q32-->qter) were observed in an abnormal boy who died at age 8 weeks of a complex cyanotic cardiac defect. He also had minor anomalies, sagittal craniosynostosis, triphalangeal thumbs, hypospadias, and a bifid scrotum. Two other infants with similar cytogenetic abnormalities were described previously. These patients had severe congenital heart defect, genitourinary anomalies, broad nasal bridge, low hairline, apparently low-set ears, short neck, and triphalangeal thumbs, in common with our patient. We suggest that combined monosomy 9p23,24-->pter and trisomy 5q31,32-->qter may constitute a clinically recognizable syndrome.

Expression and characterization of human mutant (glutamic acid304) medium-chain acyl-coenzyme A dehydrogenase in mammalian cells.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a frequent and sometimes fatal inherited metabolic disorder of fatty acid beta-oxidation. A eukaryotic expression system was used to study naturally occurring mutations in MCAD. The 1263 nucleotide coding region of human MCAD cDNA was inserted downstream of the SV40 early promoter for high-level expression in Chinese hamster ovary cells. Both normal MCAD cDNA and a mutant MCAD cDNA containing the common, disease-causing A to G transition at position 985 (A985G), which alters a lysine to a glutamic acid (K304E), were inserted into expression vectors. Transient transfection of Chinese hamster ovary cells was performed with the expression constructs. The steady state level of expressed normal MCAD protein antigen was substantially higher (5-fold) than the expressed mutant protein. The MCAD enzymatic activity in protein extracts from cells containing the expressed normal MCAD cDNA was also much higher (6-fold) than the activity in cells expressing the mutant MCAD. Therefore, these data confirm that the common K304E mutation causes MCAD deficiency primarily by decreased protein stability rather than reduction of catalytic activity and, in fact, demonstrate that the K304E mutant protein has a similar sp act against octanoyl CoA substrate as the normal protein.

Detection and assessment by positron emission tomography of a genetically determined defect in myocardial fatty acid utilization (long-chain acyl-CoA dehydrogenase deficiency).

Genetic defects in fatty acid oxidation are important, inherited causes of cardiomyopathy, skeletal myopathies, and childhood sudden death. The clinical manifestations and their severity vary widely among affected subjects and different age groups. Although measurement of serum and urinary fatty acid intermediary metabolites and enzymatic assays establish the diagnosis of a defect in fatty acid oxidation, they do not predict the specific clinical manifestations nor their severity in a given subject. To determine whether impaired myocardial fatty acid utilization, indicative of cardiac phenotypic expression of a specific genetic abnormality in fatty acid oxidation, can be detected, cardiac positron emission tomography with the metabolic tracers carbon-11-labeled palmitate and acetate was performed in 6 patients with long-chain acyl-CoA dehydrogenase (ACD) deficiency and in 9 control subjects. The myocardial extraction of both tracers was similar in patients and controls. The rate of clearance of palmitate from myocardium was significantly prolonged in patients compared with that in control subjects (0.022 +/- 0.012 vs 0.061 +/- 0.033 min-1; p < 0.025), indicative of a decreased rate of oxidation of long-chain fatty acids. Furthermore, the extent of diminution of clearance of palmitate, quantified in terms of the rate of clearance for palmitate divided by that for acetate (to correct for individual differences in overall mitochondrial oxidative metabolic flux), correlated with the clinical severity of the long-chain ACD deficiency. Accordingly, noninvasive evaluation with positron emission tomography may not only facilitate diagnosis, but also enable assessment of the pathogenetic impact and effects of therapeutic interventions in the hearts of subjects with specific, inherited defects in fatty acid oxidative metabolism.

Microcephalic osteodysplastic primordial dwarfism, type II. Report of a case with characteristic skeletal features.

Osteodysplastic primordial dwarfism, type II manifests typical skeletal features which have not previously been described in the radiographic literature. We present an infant with characteristic findings and discuss related conditions, especially the Seckel syndrome.

Inhibition of development of characteristic indices of myeloid differentiation by a factor released from a tetraploid variant of the human promyelocytic cell line, HL-60.

HL-60TR, a tetraploid variant of the human promyeloid cell line HL-60, was obtained by culturing HL-60 cells for one week with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 400 pM. HL-60TR cells have lost the ability to develop several common markers of maturation in response to compounds that induce monocytoid or myelocytoid differentiation of HL-60 cells. In addition, they release a factor which inhibits induction of the same markers in HL-60 cells. Medium conditioned by HL-60TR cells also inhibits colony formation by normal mouse bone marrow cells. These properties have been maintained by HL-60TR cells through more than one year of constant subculture in the absence of TPA, a finding which suggests the possibility that TPA may promote tumor formation not only through direct effects on the phenotype of initiated cells but also through induction of continued production of factors that affect differentiation of normal stem cells.

Effect of 5-azacytidine on differentiation and DNA methylation in human promyelocytic leukemia cells (HL-60).

One of the most readily quantitated indices of myeloid maturation in HL-60 cells is their ability to respond to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity. HL-60 cells exposed to the antileukemic drug, 5-azacytidine (3 to 5 microM) for 24 hr and subsequently cultured in its absence for 2 to 3 days develop an enhanced ability to respond to 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity detectable as an increase both in hexose monophosphate shunt activity and in the proportion of the population producing superoxide anion. 5-Azacytidine treatment also causes marked inhibition of DNA methyltransferase, and thus DNA synthesized by HL-60 cells during the 24-hr period of analogue treatment is essentially devoid of methylated cytosine residues. This suggests, as does our previous finding that a general inhibitor of transmethylation reactions, L-ethionine, can induce differentiation of HL-60 cells, that changes in gene expression triggered by these compounds may be linked to their ability to alter patterns of DNA methylation. Since at least 50% of HL-60 cells capable of forming colonies in soft agar after a 24-hr exposure to 5-azacytidine yield progeny that mature (i.e., produce superoxide anion in response to 12-O-tetradecanoylphorbol-13-acetate) 2 weeks after 5-azacytidine treatment, the results also indicate that the changes induced in HL-60 cells by limited exposure to 5-azacytidine are heritable and can influence gene expression many generations after treatment has been terminated.

L-Ethionine as an inducer of differentiation in human promyelocytic leukemia cells (HL-60).

The methionine analog, L-ethionine, induces morphological and biochemical changes in cultured HL-60 cells which are indicative of myeloid maturation. After 3 to 5 days of growth in the presence of L-ethionine, the majority of cells have enhanced phagocytic ability. The percentage of cells in the culture which bear complement receptors and which can respond to 12-O-tetradecanoylphorbol-13-acetate with respiratory burst activity increases more than 3-fold. Since the cells fail to become adherent and lose nonspecific esterase activity, we conclude that L-ethionine, like dimethyl sulfoxide, induces granulocytic differentiation of HL-60 cells.

Catabolism of 2-deoxyglucose by phagocytic leukocytes in the presence of 12-O-tetradecanoyl phorbol-13-acetate.

We have found that phagocytic leukocytes exposed to the tumor-promoting agent, 12-O-tetradecanoyl phorbol-13-acetate, efficiently release carbon-1 of 2-deoxyglucose in the form of CO2 with concurrent intracellular accumulation of a phosphorylated 5-carbon intermediate. In the absence of 12-O-tetradecanoyl phorbol-13-acetate, these cells release barely detectable amounts of CO2 from 2-deoxyglucose. 12-O-Tetradecanoyl phorbol-13-acetate, at a concentration of 1 ng/ml, has an immediate effect on CO2 release, which is temperature-dependent and linear with time and cell number. The ability of a number of phorbol ester-like compounds to enhance this catabolic pathway for 2-deoxyglucose correlates with their ability to act as tumor promotors and inflammatory agents. Although this effect of phorbol esters appears to be restricted to granulocytes, monocytes, and macrophages, the possibility arises that other mammalian cells are capable of catabolizing or can be induced to catabolize-2-deoxyglucose. Thus, 2-deoxyglucose decarboxylation should be considered whenever this analog of mannose and glucose is used as an indicator for sugar transport, especially when pharmacodynamic agents are present.

Isolation of a granulocyte colony inhibitory factor derived from human polymorphonuclear neutrophils.

An inhibitory factor obtained from mature human granulocytes which suppresses granulocyte and monocyte-macrophage colony formation by an action on the endogenous colony stimulating factor-producing cells has been partially purified and characterized. The methods for purification consisted of a combination of ultracentrifugation, DEAE-Sephadex chromatography, SDS polyacrylamide gel electrophoresis and isoelectric focusing. The material had a molecular weight range of 102--128 000 and an isoelectric point between pH 6.2 and 6.4. The inhibitory factor was found to be heat stable and glycoprotein in nature.