Addition of Anti-thymocyte Globulin in Allogeneic Stem Cell Transplantation With Peripheral Stem Cells From Matched Unrelated Donors Improves Graft-Versus-Host Disease and Relapse Free Survival.

Anti-thymocyte globulin (ATG) is commonly used to prevent graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). To evaluate the impact of ATG as part of the GvHD prophylaxis in our institution, we report the outcome of 415 patients with matched unrelated donors (MUD) transplanted for hematological malignancies with or without ATG from 2005 to 2019 at Oslo University Hospital, Norway. The following groups were compared: (1) 154 patients transplanted with peripheral blood stem cells (PBSC) without ATG 2005-2014. (2) 137 patients transplanted with bone marrow stem cells (BMSC) 2005-2019. (3) 124 patients transplanted with PBSC and ATG (PBSC + ATG) 2014-2019. Three years survival was similar in the groups, 61% following allografting with PBSC, 54% with BMSC, and 59% with PBSC + ATG. Acute GvHD grade III-IV was 14%, 14%, and 7%; chronic GvHD was 81%, 32, and 26%; and extensive cGvHD 44%, 15%, and 6% in the corresponding groups. Both acute and chronic GvHD were significantly reduced in the PBSC + ATG-versus the PBSC group (p < 0.05 and p < 0.001 respectively).Transplant-related mortality (TRM) was 33%, 25%, and 17% (p = 0.18). Graft versus host disease and relapse free survival (GRFS) at 3 years was 43 %, 43%, and 64% in the groups. Adding ATG to the GvHD prophylaxis regimen of MUD allo-HSCT with PBSC resulted in a substantial reduction of both acute and chronic GvHD without compromising the disease control, reflected in a superior 3 years GRFS.

The CD34+ Cell Dose Matters in Hematopoietic Stem Cell Transplantation with Peripheral Blood Stem Cells from Sibling Donors.

The effect of CD34+ cell dose in allogeneic hematopoietic stem cell transplantation (HSCT) on overall survival (OS) and incidence of acute and chronic graft-versus-host disease (GvHD) has not been established and few studies have been performed. Our single center analysis included 189 patients with hematological malignancies who received peripheral blood stem cell (PBSC) grafts from sibling donors. Myeloablative conditioning was used in 88 cases and 101 received reduced intensity conditioning. The median CD34+ cell dose was 5.6 × 106/kg (0.6-17.0). In the multivariate analysis, a CD34 cell dose of 6-7 × 106/kg was associated with better OS and lower transplant-related mortality (TRM), while a dose of <5 × 106/kg led to increased relapse and reduced chronic GVHD (cGVHD). A high CD34 cell-dose (>6.5 × 106/kg) correlated with less acute GVHD (aGVHD) II-IV. We conclude that the CD34 cell dose has an impact on the outcome of HSCT from sibling donor PBSCs.

Safety assessment of the biotechnologically produced human-identical milk oligosaccharide 3-Fucosyllactose (3-FL).

3-Fucosyllactose (3-FL), a highly abundant complex carbohydrate in human breast milk, functions as a prebiotic promoting early microbial colonization of the gut, increasing pathogen resistance and modulating immune responses. To investigate potential health benefits, 3-FL was produced by fermentation using a genetically modified E. coli K12 strain. The safety assessment of 3-FL included acute oral toxicity, in vitro and in vivo assessment of genetic toxicity, and a subchronic rodent feeding study. 3-FL was not acutely toxic at 5000 mg/kg bw, and there was no evidence of genetic toxicity in the bacterial reverse mutation test and chromosomal aberration assay. There was a repeatable statistically-significant trend in the 4-h S9-activated test conditions in the in vitro micronucleus assay; the confirmatory in vivo mouse micronucleus study was negative at all doses. Dietary subchronic exposure of rats to 3-FL (5% and 10%) did not produce any statistical or biologically-relevant differences in growth, food intake or efficiency, clinical observations, or clinical or anatomic pathology changes at average daily intakes of 5.98 and 7.27 g/kg bw/day for males and females, respectively. The weight of evidence from these studies support the safe use of 3-FL produced using biotechnology as a nutritional ingredient in foods.

The role of delayed bone age in the evaluation of stature and bone health in glucocorticoid treated patients with Duchenne muscular dystrophy.

BACKGROUND: Low bone mineral density and an increased risk of appendicular and vertebral fractures are well-established consequences of Duchenne muscular dystrophy (DMD) and the risk of fractures is exacerbated by long-term glucocorticoid treatment. Monitoring of endocrine and skeletal health and timely intervention in at-risk patients is important in the management of children with DMD. METHODS: As part of the Norwegian Duchenne muscular dystrophy cohort study, we examined the skeletal maturation of 62 boys less than 18 years old, both currently glucocorticoid treated (n = 44), previously treated (n = 6) and naïve (n = 12). The relationship between bone age, height and bone mineral density (BMD) Z-scores was explored. RESULTS: The participants in the glucocorticoid treated group were short in stature and puberty was delayed. Bone age was significantly delayed, and the delay increased with age and duration of treatment. The difference in height between glucocorticoid treated and naïve boys was no longer significant when height was corrected for delayed skeletal maturation. Mean BMD Z-scores fell below - 2 before 12 years of age in the glucocorticoid treated group, with scores significantly correlated with age, duration of treatment and pubertal development. When BMD Z-scores were corrected for by retarded bone age, the increase in BMD Z-scores was significant for all age groups. CONCLUSION: Our results suggest that skeletal maturation should be assessed in the evaluation of short stature and bone health in GC treated boys with DMD, as failing to consider delayed bone age leads to underestimation of BMD Z-scores and potentially overestimation of fracture risk.

Effective size of density-dependent two-sex populations: the effect of mating systems.

Density dependence in vital rates is a key feature affecting temporal fluctuations of natural populations. This has important implications for the rate of random genetic drift. Mating systems also greatly affect effective population sizes, but knowledge of how mating system and density regulation interact to affect random genetic drift is poor. Using theoretical models and simulations, we compare Ne in short-lived, density-dependent animal populations with different mating systems. We study the impact of a fluctuating, density-dependent sex ratio and consider both a stable and a fluctuating environment. We find a negative relationship between annual Ne /N and adult population size N due to density dependence, suggesting that loss of genetic variation is reduced at small densities. The magnitude of this decrease was affected by mating system and life history. A male-biased, density-dependent sex ratio reduces the rate of genetic drift compared to an equal, density-independent sex ratio, but a stochastic change towards male bias reduces the Ne /N ratio. Environmental stochasticity amplifies temporal fluctuations in population size and is thus vital to consider in estimation of effective population sizes over longer time periods. Our results on the reduced loss of genetic variation at small densities, particularly in polygamous populations, indicate that density regulation may facilitate adaptive evolution at small population sizes.

Reliability of the English version of the painDETECT questionnaire.

BACKGROUND: The painDETECT questionnaire (PD-Q) has been used widely for the identification of neuropathic pain (NeP); however, the reliability of the English version of the PD-Q has never been investigated. OBJECTIVE: This study aimed to determine the reliability of the PD-Q pre- (T0) and immediately post- (T1) clinical consultation and at one-week follow-up (T2). METHODS: We recruited 157 patients attending a Neurosurgery Spinal Clinic and Pain Management Department. Minor changes to PD-Q instructions were made to facilitate patient understanding; however, no changes to individual items or scoring were made. Intraclass correlation coefficients (ICCs) were used to assess the reliability of PD-Q total scores between T0-T1 and T0-T2; weighted kappa (κ) was used to assess the agreement of PD-Q classifications (unlikely NeP, ambiguous, likely NeP) between all time-points. To ensure stability of clinical pain, patients scoring ≤2 or ≥6 on the Patient Global Impression Scale (PGIC) at T2 were excluded from the T0-T2 analysis. RESULTS: Accounting for missing data and exclusions (change in PGIC score), data for 136 individuals (mean [SD] age: 56.8 [15.2]; 54% male) was available, of whom n = 129 were included in the T0-T1 and n = 69 in the T0-T2 comparisons. There was almost perfect agreement between the PD-Q total scores at T0-T1 time-points (ICC 0.911; 95% CI: 0.882-0.941) and substantial agreement at T0-T2 (ICC 0.792; 95% CI: 0.703-0.880). PD-Q classifications demonstrated substantial agreement for T0-T1 (weighted κ: 0.771; 95% CI: 0.683-0.858) and for T0-T2 (weighted κ: 0.691; 95% CI: 0.553-0.830). Missing data was accounted in 13% of our cohort and over 42% of our patients drew multiple pain areas on the PD-Q body chart. CONCLUSION: The English version of the PD-Q is reliable as a screening tool for NeP. The validity of the questionnaire is still in question and has to be investigated in future studies.

In vivo micronucleus studies with 6 titanium dioxide materials (3 pigment-grade & 3 nanoscale) in orally-exposed rats.

Six pigment-grade (pg) or ultrafine (uf)/nanoscale (anatase and/or rutile) titanium dioxide (TiO2) particulates were evaluated for in vivo genotoxicity (OECD 474 Guidelines) in male and female rats by two different laboratories. All test materials were robustly characterized. The BET surface areas of the pg and uf samples ranged from 7 to 17 m(2)/g and 50 to 82 m(2)/g respectively. The materials were assessed for induction of micronuclei and toxicity in bone marrow by analyzing peripheral blood reticulocytes (RETs) by flow cytometry. Single oral gavage doses of 500, 1000 or 2000 mg/kg body weight (bw) of each material were implemented with concurrent negative (water) and positive controls (cyclophosphamide). Approximately 48 and 72 h after exposure, blood samples were collected and 20,000 RETs per animal were analyzed. For each of the six tests, there were no biologically or toxicologically relevant increases in the micronucleated RET frequency in any TiO2 exposed group at either time point at any dose level. In addition, there were a lack of biologically relevant decreases in %RETs among total erythrocytes. All six TiO2 test substances were negative for in vivo genotoxicity effects; however, it is noted that the exposure to target tissues was likely negligible. One pigment grade and one ultrafine material each were evaluated for potential systemic exposure/uptake from the gastrointestinal tract by analysis of TiO2 into blood and liver. No significant increases in TiO2 over controls were measured in blood (48 or 72 h) or liver (72 h) following exposures to 2000 mg/kg bw TiO2. These data indicate that there was no absorption of the test material from the gastrointestinal tract into the blood circulation and the lack of genotoxic effects is therefore attributed to a lack of exposure due to the inability of the test material to migrate from the gastrointestinal tract into the blood and then into target tissues.

Anti-cytokine autoantibodies preceding onset of autoimmune polyendocrine syndrome type I features in early childhood.

PURPOSE: Almost all patients with autoimmune polyendocrine syndrome (APS)-I have high titer neutralizing autoantibodies to type I interferons (IFN), especially IFN-ω and IFN-α2, whatever their clinical features and onset-ages. About 90 % also have antibodies to interleukin (IL)-17A, IL-17F and/or IL-22; they correlate with the chronic mucocutaneous candidiasis (CMC) that affects ~90 % of patients. Our aim was to explore how early the manifestations and endocrine and cytokine autoantibodies appear in young APS-I patients. That may hold clues to very early events in the autoimmunization process in these patients. METHODS: Clinical investigations and autoantibody measurements in 13 APS-I patients sampled before age 7 years, and 3 pre-symptomatic siblings with AIRE-mutations in both alleles. RESULTS: Antibody titers were already high against IFN-α2 and IFN-ω at age 6 months in one sibling-8 months before onset of APS-I-and also against IL-22 at 7 months in another (still unaffected at age 5 years). In 12 of the 13 APS-I patients, antibody levels were high against IFN-ω and/or IL-22 when first tested, but only modestly positive against IFN-ω in one patient who had only hypo-parathyroidism. Endocrine organ-specific antibodies were present at age 6 months in one sibling, and as early as 36 and 48 months in two of the six informative subjects. CONCLUSION: This is the first study to collate the onset of clinical features, cytokine and endocrine autoantibodies in APS-I infants and siblings. The highly restricted early autoantibody responses and clinical features they show are not easily explained by mere loss of broad-specific self-tolerance inducing mechanisms, but hint at some more sharply focused early event(s) in autoimmunization.

Safety assessment of N-acetyl-L-threonine.

N-acetyl-L-threonine (NAT) is a dietary constituent that has been identified at low concentrations (< 1 microg/g fresh weight) in numerous foods. The current paper reports the outcome of toxicology studies conducted to assess the effects of NAT. No evidence of mutagenicity or genotoxicity was observed in in vitro bacterial or in vivo mammalian studies. No mortalities or evidence of adverse effects were observed in Sprague-Dawley (SD) rats following acute oral administration of 2000 mg of NAT/kg of body weight (kg of bw). A 28-day repeated dose toxicity study was conducted in SD rats by incorporating NAT into diets at concentrations targeting up to 1000 mg of NAT/kg of bw/day. All rats survived until scheduled sacrifice and no biologically significant differences were observed in any of the NAT treatment groups for body weights, feed consumption, clinical signs, behavioral, ophthalmology, hematology, coagulation, clinical chemistry, organ weights, or gross or microscopic changes. Based on these results, NAT does not represent a risk for mutagenicity or genotoxicity, is not acutely toxic, and the no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAT is 848.5 and 913.6 mg/kg of bw/day for male and female SD rats, respectively.

Toxicology studies with N-acetyl-L-serine.

N-Acetyl-L-serine (NAS) is a component of dietary proteins and a minor constituent of foods as a free amino acid. The current paper reports the outcome of toxicology studies conducted to assess the safety of NAS. No evidence of mutagenicity was observed in the reverse bacterial mutation assay. Genotoxicity was not observed in the bone marrow micronucleus assay conducted in mice. No mortalities or evidence of adverse effects were observed in Sprague-Dawley (SD) rats following acute oral administration at a dose of 2000 mg of NAS/kg of body weight. Similarly, no evidence of adverse effects was observed in SD rats following repeated dose dietary exposure (28-days) to targeted doses of 100, 500, or 1000 mg of NAS/kg of body weight/day. All rats survived until scheduled sacrifice and no biologically significant differences were observed in any of the response variables from the NAS exposure groups compared with untreated control groups. Based on these results, NAS does not represent a risk for mutagenicity or genotoxicity, is not acutely toxic, and the no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAS is 839.7 and 893.6 mg of NAS/kg of body weight/day for male and female rats, respectively.

Anti-inflammatory properties of enamel matrix derivative in human blood.

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro-inflammatory cytokine [tumor necrosis factor (TNF)-alpha], an anti-inflammatory cytokine (interleukin-10) and a chemokine (interleukin- 8) in whole human blood challenged by bacterial cell wall components. MATERIAL AND METHODS: Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8-(4-chlorophenyl)thio-cAMP (8-CPT-cAMP). TNF-alpha, interleukin-8 and interleukin-10 were analysed from plasma by enzyme-linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA). RESULTS: We found that EMD attenuated the release of TNF-alpha and interleukin-8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin-10 was unchanged. Enamel matrix derivative also produced a four-fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8-CPT-cAMP attenuated the formation of TNF-alpha, but not of interleukin-10, in blood challenged by lipopolysaccharide. CONCLUSION: Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti-inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells.

Cytokine responses to fungal pathogens in Kupffer Cells are Toll-like receptor 4 independent and mediated by tyrosine kinases.

Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in cytokine production, using primary cultures of rat and murine Kupffer cells exposed to Aspergillus fumigatus and Candida albicans hyphae and conidia. All fungal components induced the release of tumour necrosis factor-alpha (TNF-alpha), but with delayed kinetics compared with lipopolysaccharide (LPS). Candida albicans was the most potent inducer of TNF-alpha protein and mRNA and the only inducer of interleukin-10 (IL-10) in rat Kupffer cells. All fungal components induced enhanced mRNA levels of macrophage inhibitory protein-2 (MIP-2) in the cells, similar to LPS. Inhibitors of Src tyrosine kinases added to cells prior to stimulation led to attenuation in the release of both TNF-alpha (60%, P < 0.05) and IL-10 (70%, P < 0.05) induced by C. albicans conidia but did not influence the LPS-mediated cytokine release. Murine Kupffer cells (C57BL/10J) also released TNF-alpha as well as the chemokines keratinocyte-derived chemokine (KC) and MIP-2 in response to fungal component. Surprisingly, Kupffer cells from TLR4-deficient C57BL/ScCr mice exhibited significantly enhanced production of KC and MIP-2 upon stimulation by fungal components compared with control littermates (P < 0.05). Our study demonstrates that Aspergillus and Candida components induce cytokine production in rat Kupffer cells and that the response to C. albicans conidia involves Src tyrosine kinases. The experiments with TLR4-deficient Kupffer cells suggest that the cytokine response in these cells to fungal component is not mediated by TLR4.

Altered composition of HDL3 in FH subjects causing a HDL subfraction with less atheroprotective function.

BACKGROUND: Subjects with familial hypercholesterolemia (FH) are associated with increased risk of premature atherosclerosis and coronary artery disease (CAD). However, onset of clinically manifested CAD varies widely among subjects with heterozygous FH. The purpose of this study was to investigate whether FH subjects with an identical mutation in the low-density lipoprotein (LDL) receptor gene have a high-density lipoprotein (HDL)3 that is characterized by a less atheroprotective functions than that of healthy controls and within subgroups of FH. DESIGN: Twenty-two adults <75 years of age with FH and 17 healthy sex- and age-matched controls were included. HDL3 was isolated and the composition was characterized from each subject, and its ability to suppress tumor necrosis factor(TNF)-alpha stimulated expression of ICAM-1 on HUVEC was investigated. In addition, plasma level of soluble sICAM-1 and VCAM-1 was measured. RESULTS: Compared to controls, FH subjects had lower content of phospholipids in their HDL3 subfraction and a higher serum ICAM-1 level. No differences in sVCAM-1 were observed. HDL3 isolated from FH with body mass index(BMI)>25 and from FH subjects with premature CAD contained higher content of triglycerides compared to the HDL3 from FH subjects with BMI<25 and without CAD, respectively. Most important, when testing the function of HDL3 in the two FH subgroups characterized by elevated BMI and premature CAD, lower inhibition of ICAM-1 expression on HUVEC was observed. CONCLUSIONS: The altered composition of HDL3 from FH subjects with BMI>25 and FH subjects with premature CAD may be responsible for a HDL3 subfraction with less protective properties assessed as inhibition of ICAM-1 expression on HUVEC consequently leading to more proatherogenic endothelial surface.

High frequency of coeliac disease among patients with autoimmune adrenocortical failure.

BACKGROUND: Coeliac disease (CD) is an autoimmune disease of the small intestine caused by gluten ingestion in genetically predisposed subjects. It can occur isolated or in combination with other autoimmune diseases. Autoimmune Addison's disease is frequently associated with other organ-specific autoimmune diseases. We have investigated the prevalence of CD among a large cohort of patients with autoimmune Addison's disease. METHODS: Seventy-six patients (44 women) with Addison's disease, 52% of whom had polyendocrine failure, were recruited from a registry of organ-specific autoimmune diseases in Norway. All sera were analysed for antibodies against gliadin (AGA), endomysium (EMA) and tissue transglutaminase (tTG). Patients with positive EMA and/or anti-tTG were offered endoscopy. The human leucocyte antigen (HLA) class II genotypes were determined. RESULTS: Five patients had antibodies against both endomysium and tissue transglutaminase. In these five patients, CD was verified by biopsy. One patient had known CD prior to the study. All six patients with CD carried the CD-associated HLA haplotype DR3-DQ2. The total prevalence of CD was 7.9%. CONCLUSION: CD is frequently associated with Addison's disease. The risk of developing CD seems to be higher than can be explained by the common DR3-DQ2 association alone. It is often asymptomatic or associated with unspecific symptoms. Addison patients should be screened for the presence of CD on a regular basis.

Anogenital human papillomavirus in non-abused preschool children.

AIM: To estimate the prevalence of human papillomavirus (HPV) in anogenital samples from children selected for non-abuse. METHODS: A letter of invitation was sent to 2731 girls and 1042 boys, all of them aged 5 or 6 y. Inclusion was based on self-selection, whereby parents who did not suspect any occurrence of sexual abuse of their child gave informed consent to participate. Several mechanisms were undertaken to exclude abused children. A complete examination was done of each child, including anogenital examination with a colposcope and microbiological sampling from the genitals and anus. Polymerase chain reaction (PCR) using primers MY09 and MY11 was used to identify HPV, and sequencing was done on each positive amplicon. RESULTS: PCR was performed on 325 adequate specimens from 211 children enrolled. Seven samples from 5 girls were HPV-positive, making 2/161 (1.2%) of the anal and 5/164 (3.0%) of the genital specimens positive. HPV was not detected in any of the boys. In four girls strong associations with HPV 6 genotypes were found, while one girl probably had a mixed infection with HPV 6 and 16. Three girls (1.8%) had clinically detectable anogenital warts. CONCLUSION: Since our results are comparable with a prevalence reported from allegedly abused children, and higher rates have been reported from the oral cavity in healthy children, we find detection of HPV unreliable as an indicator of sexual abuse in 5-6-y-old children. The rate of anogenital warts found in our study is comparable with a rate reported in abused children.

Genital anatomy in non-abused preschool girls.

AIM: To describe the normal variations in genital anatomy in preschool girls selected for non-abuse. METHODS: A total of 2731 girls aged 5 or 6 y were invited to take part in the study; 195 girls were recruited. Inclusion was based on self-selection, whereby parents who did not suspect any occurrence of sexual abuse of their children gave informed consent to participate. Several steps were taken to exclude abused girls and girls with previous accidental genital injuries. The genital examination, using a colposcope and a camera, was performed in supine position using a separation and traction technique, and in the prone knee-chest position. RESULTS: A number of genital anatomical features and hymenal measurements were described and found consistent with previous studies. An important finding was outward folding of the posterior hymenal rim in many girls, a feature that could be difficult to distinguish from attenuation of the posterior hymen. A gaping hymenal orifice, previously suggested to be a supportive sign of sexual abuse, was fairly frequently found and significantly associated with a large horizontal hymenal diameter. CONCLUSION: To distinguish between girls with outward folding of the posterior hymen and those with attenuated hymens, we recommend the use of the saline irrigation method. Even though normative hymenal measurement data now exist from a reasonable number of girls, these measurements should be used with caution in sexual abuse evaluations.

Anogenital bacteriology in non-abused preschool children: a descriptive study of the aerobic genital flora and the isolation of anogenital Gardnerella vaginalis.

The purpose of the study is to describe the genital aerobic bacterial flora including Gardnerella vaginalis in girls and the occurrence of anal G. vaginalis in both genders. From a group of 3773 children, 278 (99 boys and 179 girls) with a mean age of 5.63 y (range: 5.13-6.73) were recruited. Inclusion in the study was based on self-selection, whereby parents who did not suspect any occurrence of sexual abuse of their child gave informed consent to participate. Several mechanisms were undertaken to exclude abused children. At least one bacterial species was isolated from the genitals of 59 (33.9%) girls. Most isolates (39 out of 99) were bacteria representing skin flora (staphylococci and coryneform organisms), with viridans streptococci and related organisms as the second most common group of isolates (31 out of 99). S. anginosus was the single most frequent bacterial species identified (17 isolates). Streptococcus pyogenes was isolated from the genitals of two girls, Streptococcus pneumoniae from one girl and Haemophilus influenzae from eight girls. G. vaginalis was not isolated from the genitals in any girl, but the organism was isolated from the anal canal in three children.

Analysis of short stature homeobox-containing gene ( SHOX) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity.

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.

Twelve novel mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL) in patients with various forms of hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report here the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 11 families affected by various forms of hypophosphatasia. Nineteen distinct mutations were found, 7 of which were previously reported. Eleven of the 12 new mutations were missense mutations (Y11C, A34V, R54H, R135H, N194D, G203V, E218G, D277Y, F310G, A382S, V406A), the last one (998-1G>T) was a mutation affecting acceptor splice site.