Prolonged Disease Course of COVID-19 in a Patient with CTLA-4 Haploinsufficiency.

Patients with primary immunodeficiencies are especially vulnerable to developing severe coronavirus disease 2019 (COVID-19) after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an important regulator of immune responses, and patients who suffer from CTLA4 haploinsufficiency have hyperactivation of effector T cells and infiltration of various organs. Overexpression of CTLA4 has been associated with a more severe disease course in patients with COVID-19, but there have only been a few reports on the disease course of COVID-19 in patients with CTLA4 haploinsufficiency. We report on a 33-year-old female with a history of immune thrombocytopenia, autoimmune haemolytic anaemia, granulomatous-lymphocytic interstitial lung disease, and common variable immunodeficiency who developed COVID-19. She was admitted and discharged from the hospital several times in the months thereafter and remained symptomatic and had a positive SARS-CoV-2 PCR for up to 137 days after the first symptoms. No SARS-CoV-2 antibodies were identified in the patients' serum. The disease was finally controlled after repeated infusions of convalescent plasma and treatment of concurrent bacterial and fungal infections. Genetic analysis revealed a likely pathogenic variant in CTLA4, and CTLA4 expression on regulatory T-cells was low. This case illustrates that patients with primary immunodeficiencies who have a protracted disease course of COVID-19 could benefit from convalescent plasma therapy.

The quantum dynamics of H2 on Cu(111) at a surface temperature of 925 K: Comparing state-of-the-art theory to state-of-the-art experiments 2.

State-of-the-art 6D quantum dynamics simulations for the dissociative chemisorption of H2 on a thermally distorted Cu(111) surface, using the static corrugation model, were analyzed to produce several (experimentally available) observables. The expected error, especially important for lower reaction probabilities, was quantified using wavepackets on several different grids as well as two different analysis approaches to obtain more accurate results in the region where a slow reaction channel was experimentally shown to be dominant. The lowest reaction barrier sites for different thermally distorted surface slabs are shown to not just be energetically, but also geometrically, different between surface configurations, which can be used to explain several dynamical effects found when including surface temperature effects. Direct comparison of simulated time-of-flight spectra to those obtained from state-of-the-art desorption experiments showed much improved agreement compared to the perfect lattice BOSS approach. Agreement with experimental rotational and vibrational efficacies also somewhat improved when thermally excited surfaces were included in the theoretical model. Finally, we present clear quantum effects in the rotational quadrupole alignment parameters found for the lower rotationally excited states, which underlines the importance of careful quantum dynamical analyses of this system.

The quantum dynamics of H2 on Cu(111) at a surface temperature of 925 K: Comparing state-of-the-art theory to state-of-the-art experiments.

We present results of our recently expanded static corrugation model (SCM) approach that included the relevant surface temperature effects, applied to the dissociative chemisorption reaction of H2 on a Cu(111) surface. The reaction and rovibrationally elastic scattering probabilities that we obtain at a quantum dynamical (QD) level, as an average of many statically distorted surface configurations, show great agreement with those of a dynamic surface model, which reinforces the validity of the sudden approximation inherent to the SCM. We further investigate several simple methods of binning the final rovibrational state of quasi-classical dynamics simulations, to find those best suited to reproduce the QD results for our system. Finally, we show that the SCM obtained results reproduce experimental dissociation curves very well, when the uncertainty in experimental saturation values is taken into account. Some indication of a slow channel, so far only observed in experiment, can also be found at low incidence energies, although more rigorous QD simulations are required to reduce the noise inherent to our propagation methods.

Accurate description of the quantum dynamical surface temperature effects on the dissociative chemisorption of H2 from Cu(111).

Accurately describing surface temperature effects for the dissociative scattering of H2 on a metal surface on a quantum dynamical (QD) level is currently one of the open challenges for theoretical surface scientists. We present the first QD simulations of hydrogen dissociating on a Cu(111) surface, which accurately describe all relevant surface temperature effects, using the static corrugation model. The reaction probabilities we obtain show very good agreement with those found using quasi-classical dynamics (QCD), both for individual surface slabs and for an averaged, thus Monte Carlo sampled, set of thermally distorted surface configurations. Rovibrationally elastic scattering probabilities show a much clearer difference between the QCD and QD results, which appears to be traceable back toward thermally distorted surface configurations with very low dissociation probabilities and underlines the importance of investigating more observables than just dissociation. By reducing the number of distorted surface atoms included in the dynamical model, we also show that only including one surface atom, or even three surface atoms, is generally not enough to accurately describe the effects of the surface temperature on dissociation and elastic scattering. These results are a major step forward in accurately describing hydrogen scattering from a thermally excited Cu(111) surface and open up a pathway to better describe reaction and scattering from other relevant crystal facets, such as stepped surfaces, at moderately elevated surface temperatures where quantum effects are expected to play a more important role in the dissociation of H2 on Cu.

Beyond the static corrugation model: Dynamic surfaces with the embedded atom method.

The D2 on Cu(111) system has for many years been one of the major benchmark systems for surface scientists. Generating surface configurations using the embedded atom method (EAM), we investigate the quality of the chemically accurate static corrugation model (SCM) for including surface temperature effects, with a focus on the random displacement approach to its distorted surface generation. With this EAM potential, we also treat the Cu(111) surface of our system fully dynamically and shine a further light on not only the quality of the SCM sudden approach but also the limited effect of energy exchange with the surface. Reaction and (in)elastic scattering probability curves, as well as simulated time-of-flight spectra, show good agreement with both earlier works and experimental results, with surface reactions showing a preference for surface atoms displaced away from the incoming molecule. The good agreement with the non-static surface model also further establishes the limited effect of energy exchange on not only the reaction but also on the elastic and inelastic scattering probabilities, even though some molecular translational energy is deposited into the surface.

The yield of diagnostic work-up of patients presenting with myalgia, exercise intolerance, or fatigue: A prospective observational study.

Myalgia, fatigue, and exercise intolerance are cause for referral to a neurologist. However, the diagnostic value of history, neurological examination, and ancillary investigations in patients with these symptoms is unknown. This study provides a sound footing for deciding which ancillary investigations should be conducted. A prospective observational study of the diagnostic approach in 187 patients with myalgia, exercise intolerance, or fatigue as their predominant symptom was performed. The primary outcomes were independent contribution of referral letter, history, examination, and ancillary investigations to a myopathy diagnosis. The secondary outcome was diagnostic value of combined ancillary investigations. 27% of patients had a myopathy. Positive family history (OR 3.2), progressive symptoms (OR 2.2), atrophy (OR 9.7), weakness (OR 10.9), and hyporeflexia (OR 4.4) were associated with a myopathy. Positive predictive values for myopathy were calculated for CK (0.32), EMG (0.66), ultrasound (0.47), and muscle biopsy (0.78). All contributed significantly in predicting myopathy. Multivariate analysis yielded a diagnostic algorithm facilitating a more efficient work-up in future patients. CK levels, EMG, ultrasound, and muscle biopsy independently contribute to predicting a myopathy. The diagnostic algorithm shows which combination of ancillary investigations should be employed in different subgroups and when to omit invasive techniques. This algorithm may drastically improve diagnostic efficiency.

Detection and identification of mycobacteria in fixed stained smears and formalin-fixed paraffin-embedded tissues using PCR.

OBJECTIVE: To determine the feasibility of using polymerase chain reaction to amplify DNA from methanol-fixed, Romanowsky-stained and Ziehl-Neelsen-stained smears to confirm the presence of mycobacteria. MATERIALS AND METHODS: Tissue was obtained from 10 archival slides and 27 slides from a prospective series of consecutive cases. Phosphate buffered saline (500 µL) was pipetted onto a stained smear (on a glass slide) using a disposable filtered pipette tip. The material adherent to the slide was scraped from its surface and drawn up into the saline. Routine DNA extraction and purification was carried out before nested polymerase chain reaction testing targeting the 16S-23S internal transcribed spacer region or a TaqMan real-time polymerase chain reaction. The real-time polymerase chain reaction was also used on thick sections cut from formalin-fixed paraffin-embedded tissue blocks from 24 canine leproid granulomas. RESULTS: Mycobacterial DNA was detected in 34 of 37 slides. Polymerase chain reaction products could not be amplified from three archived smears stained using the Ziehl-Neelsen acid-fast method, probably because its harsher fixation damaged the DNA. With the nested polymerase chain reaction, species identification using internal transcribed spacer sequence analysis was achieved in all instances, diagnosing a wide range of mycobacteria. The real-time polymerase chain reaction detected Mycobacterium sp. CLG DNA within all 24 formalin-fixed paraffin-embedded specimens tested. CLINICAL SIGNIFICANCE: This technique should provide a non-invasive and cost-effective means of diagnosing mycobacterial infections.

Sleep disturbances in chronic progressive external ophthalmoplegia.

BACKGROUND: Chronic progressive external ophthalmoplegia (CPEO) is a relatively common mitochondrial disorder. In addition to extraocular muscle weakness, various other organs can typically be affected, including laryngeal and limb muscles, cerebrum, cerebellum, and peripheral nerves. Given this multi-organ involvement, patients are likely to be prone to sleep disturbances. Here, we determined the nature, prevalence, and determinants of sleep disturbances in CPEO. METHODS: We used validated questionnaires for various sleep disorders and possible determinants such as mood and anxiety, and we performed ambulant polysomnography (PSG) in 20 patients with genetically confirmed CPEO. RESULTS: Three quarters of patients reported nocturnal sleep dysfunction. Thirty-five percent of patients fulfilled the criteria for restless legs syndrome, 30% excessive daytime sleepiness, and 70% significant periodic limb movements. PSG recordings revealed several indicators of a disrupted sleep architecture. Obstructive sleep disordered breathing was present in only one patient. However, four patients had an increased central sleep apnea index, all of whom had a polymerase gamma-1 mutation and a SANDO phenotype (sensoric atactic neuropathy, dysarthria, ophthalmoplegia). Physical examination and questionnaire outcomes were poor predictors of PSG results. CONCLUSION: Several specific sleep disturbances are part of the phenotype of CPEO. Given that the disease is otherwise incurable, symptomatic treatment of sleep disturbances may be an important tool to improve quality of life. Therefore, patients with CPEO should be actively screened for sleep disorders, with a low threshold to perform PSG.

Cerebellar ataxia with elevated cerebrospinal free sialic acid (CAFSA).

In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)--a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients' urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).

[Mitochondrial diseases; thinking beyond organ specialism necessary]. / Mitochondriale ziekten; orgaanspecialisme-overstijgend denken gevraagd.

Mitochondrial disorders are caused by a defect in intracellular energy production. In general, these are multi-system disorders, predominantly affecting organs with high energy requirements. Due to the fact that mitochondrial disorders are not as rare as is generally assumed, and due to the diversity of symptoms, many different medical specialists will at some time be confronted with these patients. Early recognition ofa mitochondrial disorder reduces patient anxiety and avoids unnecessary ancillary investigations and potentially hazardous treatments. A mitochondrial disease should be considered in the event of dysfunction of more than 2 organ systems or processes with high energy requirements, certainly if there is a positive maternal family history. If fatigue includes exercise-induced muscle pain or muscle weakness, and if muscle pain predominantly occurs during exertion, a mitochondrial disease should be considered. The combination ofdiabetes mellitus and deafness is also a strong indicator of mitochondrial disease. An extensive family history should always be taken. In adults, the most frequently occurring mitochondrial syndromes are chronic progressive external ophthalmoplegia (CPEO), maternally inherited diabetes and deafness syndrome (MIDDS) and Leber's hereditary optic neuropathy. Since much research effort is currently being invested in the development of causal medical treatments, the importance of an early diagnosis is likely to become of increasing importance in the future.

Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system.

Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT-/-) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT-/- rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

Identifying polymorphisms in the Rattus norvegicus D3 dopamine receptor gene and regulatory region.

The D(3) dopamine receptor has been implicated in several neuropsychiatric disorders, including schizophrenia, Parkinson's disease and addiction. Sequence variation in the D(3) gene can lead to subtle alteration in receptor structure or gene expression and thus to a different phenotype. In this study we examine the sequence variation in the D(3) gene in 96 rat strains and substrains. Interestingly, the analyses revealed 10 polymorphisms in the 5'flanking region and four polymorphisms in intronic regions of the gene. Moreover, two single nucleotide polymorphisms (SNPs) that result in amino acid changes were found in the last exon of the D(3) gene in the RNU/Mol strain. Additionally, bioinformatic analysis of the 5'flanking region and first intron of the gene revealed putative transcription factor binding sites that are conserved between mouse and human and are affected by the SNPs, possibly resulting in altered regulation of the subsequent transcription factor.

Feline paraneoplastic syndrome associated with thymoma.

CASE HISTORY: A 6-year-old, spayed, female, domestic short-haired cat presented with severe erythroderma and scaling skin. She showed disturbed gait and mild behavioural changes. CLINICAL FINDINGS: The cat had a generalised, erythematous, scurfy dermatitis with marked, multifocal crusting and skin thickening. The skin was painful and contracted, which appeared to prevent normal freedom of movement. DIAGNOSIS: The cat was suspected to have a paraneoplastic syndrome. A mediastinal mass was located and histologically confirmed as thymoma. The cat was diagnosed with a thymoma- associated cutaneous paraneoplastic syndrome. CLINICAL RELEVANCE: This is a rare condition with few reports in the literature. The skin changes, both grossly and histologically, were considered to be different from those described in cases of paraneoplastic dermatosis associated with pancreatic neoplasia. The clinical presentation was characteristic and more cases may occur in practice than are recognised. In this case, as in previous reports, the tumour was grossly resectable, which could lead to cure of the clinical condition.

Hypogammaglobulinaemia: cumulative experience in 49 patients in a tertiary care institution.

In this paper, clinical data of 49 adult patients with agammaglobulinaemia (syn. hypogammaglobulinaemia), 15 cases of X-linked agammaglobulinaemia (XLA) and 34 of common variable immunodeficiency (CVID) are reviewed. Although immunoglobulin substitution largely abolished life-threatening respiratory tract infections, considerable infectious and non-infectious morbidity was still encountered in these patients. Almost all patients suffered from chronic or recurrent upper and lower airway infections, mainly caused by Haemophilus influenzae and pneumococci. The lower respiratory tract infections led to cumulative damage to the respiratory tract, especially in XLA patients. Also the incidence of infections outside the respiratory tract (giardiasis, Campylobacter jejuni infections) was more common in XLA patients than in CVID patients. Nodular lymphoid hyperplasia was only found in CVID. A variety of other non-infectious complications were seen especially in CVID. Neoplastic complications occurred in nine patients (two cases of thymoma, two colorectal cancer, one gastric carcinoma, two haematological malignancies, two cases of skin cancer). Six patients died (five XLA patients and one CVID patient, from infectious and non-infectious causes).

Use of interreflection and shadow for surface contact.

The interaction of light with surfaces results in a number of lighting effects that may serve as valuable visual cues. Previous research on shadows has shown them to be effective in determining the three-dimensional (3-D) layout of a scene, but interreflections have been ignored as cues for spatial layout. Interreflections as well as shadows may help to disambiguate the 3-D layout of objects by providing information about an object's contact with a surface. We generated computer images of a box on an extended textured ground plane that was either in contact with the ground or was slightly above the ground. Images were rendered for four conditions: (1) no shadow + no interreflection, (2) shadow only, (3) interreflection only, and (4) shadow + interreflection. A photometrically incorrect condition was also included. The participants rated the degree of contact for each image on a scale, which was used to generate receiver operating characteristic (ROC) curves and a measure of sensitivity. In the images with no shadows or interreflections, the participants performed at chance levels. Interreflections, shadows, and a combination of interreflections and shadows all resulted in high sensitivity for judging object contact. More important, information from shadows and interreflections can be combined, resulting in near-perfect judgment of surface contact. Interreflections and shadows can be effective cues for object contact.

Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.

Effects of source of starch on net portal flux of glucose, lactate, volatile fatty acids and amino acids in the pig.

The ileal digestibilities of maize starch and native pea starch do not differ. However maize starch is digested faster than pea starch and the ileal amino acid digestibility of a diet containing pea starch is lower. In the present study, the net portal fluxes of glucose, lactate, volatile fatty acids (VFA) and amino acids were measured for diets including 650 g maize starch or pea starch/kg. The diets were fed at a level 870 kJ digestible energy/kg0.75 twice daily (06.00 and 18.00 hours) to four female pigs in a crossover design. Portal vein blood flow did not differ between maize and pea starches (1620 and 1484 ml/min respectively; SED 100; P = 0.23). For maize starch portal glucose flux was significantly higher during the first 6 h after feeding, was not different 8 h after feeding and was significantly lower thereafter. Net portal glucose flux was higher for maize starch than for pea starch (1759 and 1265 mmol/12 h respectively; SED 182; P = 0.054). Net portal lactate flux was not significantly different between maize and pea starches (36.5 and 67.2 mmol/12 h respectively; SED 24.1; P = 0.27) and net portal VFA flux was lower for maize starch than for pea starch (169 and 218 mmol/12 h respectively; SED 18; P = 0.054). Net portal fluxes of valine, isoleucine, phenylalanine, tryptophan, arginine, serine, cystine, tyrosine, lysine, histidine and the sum of essential amino acids tended to be or were higher (P < 0.1 or P < 0.05) and net portal flux of aspartic acid tended to be lower for pea starch (P < 0.1). It can be concluded that, although ileal digestibility of both starches is equal, the rate of appearance of glucose in the portal vein was higher for maize starch, influencing the net portal flux of amino acids.

Killer cell receptors: keeping pace with MHC class I evolution.

NK cells express receptors that bind to polymorphic determinants of MHC class I heavy chains. MHC ligands vary greatly between mammalian species, and the use of distinct molecular families of NK cell receptors by humans and mice suggests that the receptors too can be evolving rapidly. The KIR (killer cell inhibitory receptor) family of receptors are found in primates and recognize class I epitopes that are of relatively recent origin in primate evolution. Therefore, KIR molecules have probably evolved class I receptor function more recently than C-type lectins, which are represented in both humans and mice. Individual humans express NK cell receptors for which they have no class I ligand, demonstrating a looseness in the coupling of expression between the receptors and their ligands. However, study of a single donor suggests that every NK cell expresses at least one inhibitory receptor for a self-HLA class I allotype, consistent with the missing self hypothesis. Thus the NK-cell receptor-class I interaction appears to control the NK-cell repertoire during ontogeny of the individual and has the potential to be a selective factor influencing both MHC class I and NK cell receptor diversity in the evolution of populations and species.

Fragile X founder effects and new mutations in Finland.

The apparent associations between fragile X mutations and nearby microsatellites may reflect both founder effects and microsatellite instability. To gain further insight into their relative contributions, we typed a sample of 56 unrelated control and 37 fragile X chromosomes from an eastern Finnish population for FMR1 CGG repeat lengths, AGG interspersion patterns, DXS548, FRAXAC1, FRAXE and a new polymorphic locus, Alu-L. In the controls, the most common FMR1 allele was 30 repeats with a range of 20 to 47 and a calculated heterozygosity of 88%. A strong founder effect was observed for locus DXS548 with 95% of fragile X chromosomes having the 21 CA repeat (196 bp) allele compared to 17% of controls, while none of the fragile X but 69% of controls had the 20 repeat allele. Although the FRAXAC1 locus is much closer than DXS548 to FMR1 (7 kb vs. 150 kb), there was no significant difference between fragile X and control FRAXAC1 allele distributions. The FRAXE repeat, located 600 kb distal to FMR1, was found to show strong linkage disequilibrium as well. A newly defined polymorphism, Alu-L, located at approximately 40 kb distal to the FMR1 repeat, showed very low polymorphism in the Finnish samples. Analysis of the combined loci DXS548-FRAXAC1-FRAXE showed three founder haplotypes. Haplotype 21-19-16 was found on 27 (75%) of fragile X chromosomes but on none of controls. Three (8.4%) fragile X chromosomes had haplotypes 21-19-15, 21-19-20, and 21-19-25 differing from the common fragile X haplotype only in FRAXE. These could have arisen by recombination or from mutations of FRAXE. A second haplotype 21-18-17 was found in four (11.1%) fragile X chromosomes but only one (1.9%) control. This may represent a more recent founder mutation. A third haplotype 25-21-15, seen in two fragile X chromosomes (5.6%) and one (1.9%) control, was even less common and thus may represent an even more recent mutation or admixture of immigrant types. Analysis of the AGG interspersions within the FMR1 CGG repeat showed that 7/8 premutation chromosomes lacked an AGG whereas all controls had at least one AGG. This supports the hypothesis that the mutation of AGG to CGG leads to repeat instability and mutational expansion.