The Master of Catherine of Cleves is considered the finest and most original illuminator of the medieval northern Netherlands, and this manuscript is his masterpiece. In 1471 Catherine watched in horror as Arnold secured his freedom and regained his title while Adolf was imprisoned. Arnold died in 1473.
Isaiah already has enough problems in his life without adding a new one-his mate. The man Fate chose for Isaiah isn't just any man, no. It's the man who kidnapped Jamie for the Glass Research Company, the man Dominic kept locked up for a year. And now he's out and living with the pride, eating with them and sleeping in the room next to Isaiah's.
John knows he's lucky. Dominic could have killed him for what he did, but the lion shifter gives him a second chance instead, and John isn't about to waste it. He's not all that comfortable around shifters yet, but it's not like he has to like them.
Even if he finds out he's a shifter's mate. As if things weren't already difficult enough, Isaiah has to go home and leave John behind. Will John come and help when Isaiah's house is attacked? Will Isaiah finally realize he can't live his life for his mother? And even if he does, will John take him back?
Subjects Subjects with a clinical diagnosis of AGS from 127 independent pedigrees were recruited internationally through collaborating physicians and the. Twenty-five families were seen by one of us (Y.J.C.).
For the remainder, information was provided by the clinician responsible for the care of the patient. Clinical, neuroimaging, and laboratory data were obtained from medical records, magnetic resonance imaging (MRI), and CT scans. Information about every clinical characteristic was not available for all patients. All mutation-negative families and those families in which only one mutation could be identified included at least one child fulfilling the following diagnostic criteria: neurological features of an encephalopathy, intracranial calcification, negative investigations for common prenatal infections, a CSF white-cell count (WCC) ⩾5 white cells/mm 3, and/or raised levels of IFN-α (2 IU/liter or 10 pg/ml) in the CSF. With consent, blood samples were obtained from affected children, their parents, and unaffected siblings.
The study was approved by a U.K. Multicentre Research Ethics Committee (reference number 04:MRE00/19). Mutation Analysis Genomic DNA was extracted from peripheral-blood leukocytes by standard methods.
PCR amplification of all coding exons and conserved splice sites of TREX1 ( AGS1), RNASEH2A ( AGS4), RNASEH2B ( AGS2), and RNASEH2C ( AGS3) was performed (primer sequences available on request). Purified PCR-amplification products were sequenced using dye-terminator chemistry and were electrophoresed on ABI 3700 (Applied Biosystems), ABI 3100, or MegaBace500 (Amersham Pharmacia) capillary sequencers. Additionally, high-resolution melting curve analysis (MCA) was employed to screen RNASEH2A and RNASEH2B, with use of a LightScanner (Idaho Technology) operating LightScanner v1.0.364 software. PCR incorporated the fluorescent dye LCGreen+, and, after completion, microtiter plates were transferred to the LightScanner, with fluorescence data collection over the temperature range 70°C–95°C, as samples were melted. Samples were run both “neat” and “spiked” with wild-type PCR product, to ensure detection of homozygous variants. Data were analyzed using the “manual scanning” setting.
For DNA fragments with more than one melting domain, as well as examination of the entire melting curve, each separate melting domain was analyzed individually. Samples in which the melting curve deviated from the wild-type control were subjected to DNA sequencing.
Mutations were classified as “null” if they were predicted to result in premature protein termination (including frameshifts, nonsense mutations, and mutations altering splice-donor and -acceptor sequences); missense mutations were defined as “those causing the substitution of one amino acid for another.” One hundred European control alleles were sequenced for all four genes, and, in the case of RNASEH2C, 100 South Asian alleles were also analyzed. Genetic Findings Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in 31, 3, 47, and 18 families, respectively. In three families, we could identify only a single RNASEH2B mutation. In one family, we identified a single mutation in RNASEH2A, and, in one further patient, we saw two putative RNASEH2A mutations on the same allele.
In one patient, a single de novo heterozygous TREX1 mutation was observed. Additionally, in 22 families, no mutations were found ( and ). None of the identified mutations was annotated as a SNP in the available databases, and none was present in alleles from control subjects. Where tested, mutations segregated with the disease in all families, and, except in the case of the de novo heterozygous TREX1 mutation, all available parents were heterozygous for a single mutation.
TREX1 is a single-exon gene encoding a 314-aa protein. Mutations were seen throughout the gene. Eleven mutations were null alleles. Eighteen families, 14 of which were of northern European origin, were homozygous (15) or compound heterozygous (3) for a c.341G→A transition (R114H). Notably, all R114H homozygotes were also homozygous for the T allele of a SNP at c.531.
This allele exhibits highly significant (χ 2 test P. Schematic representation of the TREX1 protein, with the corresponding position of the mutations identified in TREX1.
Regions 1, 3, and 4 represent the exonuclease regions (Exo1-3), which coordinate the binding of 2 Mg 2+ ions required for catalysis. Region 2 represents the polyproline II motif. Region 5 represents the dileucine-repeat region.
Numbers in parentheses after mutations represent the number of mutated alleles identified. An asterisk (.) denotes the de novo mutation identified in one family. A total of 20 distinct mutations were identified in RNASEH2B (a 308-aa protein). All were missense changes, except for five splice acceptor/donor mutations and two stop mutations; the 12 affected individuals carrying these were all compound heterozygotes; the second mutation was a missense change.
All 50 families with a change identified in RNASEH2B harbored at least one mutation in exon 2, 6, or 7. The recurrent c.529G→A (A177T) mutation was seen in a panethnic cohort of patients, indicating that this is a mutation hotspot.
In three families, we could identify only a single RNASEH2B mutation. In one of these families, the mutation had been maternally inherited, whereas, in the other two families, parental samples were unavailable. The two heterozygous mutations observed in these three pedigrees, c.529G→A and c.488C→T, were seen recurrently in other cases. Schematic representation of the RNASEH2B gene, with the position of identified mutations. Shaded areas with large numbers indicate the specified exons.
Numbers in parentheses after mutations represent the number of mutated alleles identified. Splice-site variants and stop mutations always occur with a missense mutation. We identified six distinct RNASEH2C (164-aa) missense mutations in 18 families. Thirteen of these families were of Pakistani origin, all of whom were homozygous for the R69W (c.205C→T) mutation on a common haplotype (data not shown), suggesting an ancient founder effect. The remaining mutations were seen in a panethnic cohort of patients. Schematic representation of the RNASEH2C gene, with the position of identified mutations. Shaded areas with large numbers indicate the specified exons.
Numbers in parentheses after mutations represent the number of mutated alleles identified. Four children from three families were found to have biallelic mutations in RNASEH2A (299 aa). Four of five mutations, one of which occurred in homozygous form, were missense. In one family, we identified a single mutation, c.704G→A (R236Q), in RNASEH2A that had been inherited. In another patient, we identified two putative RNASEH2A mutations, c.717dup GC and c.719C→T (T241M), on the same allele. Parental samples were unavailable in this case. Finally, a number of polymorphisms were also identified in the RNASEH2A gene.
Schematic representation of the RNASEH2A gene, with the position of identified mutations and polymorphisms. Shaded areas with large numbers indicate the specified exons. Numbers in parentheses after mutations represent the number of mutated alleles identified. An asterisk (.) denotes polymorphisms included in the SNP database; a double asterisk (.) denotes synonymous changes not found in controls or annotated as a SNP; ¶ denotes polymorphisms found in controls; † denotes polymorphisms found in combination with RNASEH2B mutations; ‡ denotes putative mutation found in patients with only a single identified RNASEH2A change; a number sign (#) denotes mutations found on one allele in a single patient. Clinical Findings Clinical data were collected for 123 individuals from 94 families with mutations in TREX1, RNASEH2A, RNASEH2B, or RNASEH2C.
The majority of children were born at term, with a normal birth weight and head circumference, and were discharged to home soon after delivery. However, 23 children (19 with TREX1 mutations, 3 with RNASEH2C mutations, and 1 with a homozygous RNASEH2A mutation) were affected at birth and required immediate support. These children presented with abnormal neurology, acted jittery, and fed poorly.
Eight experienced neonatal seizures, and 15 demonstrated liver involvement, with hepatosplenomegaly and/or raised transaminase levels. Fifteen children had a thrombocytopenia, of whom five required one or more platelet transfusions.
CReceived transfusion of platelets. All other children presented at variable periods beyond the first few days of life with, in the majority of cases, the stereotypical subacute onset of a severe encephalopathy characterized by irritability, inconsolable crying, intermittent sterile pyrexias (40%), and a loss of skills. These episodes usually lasted several months, beyond which time the condition stabilized. Thereafter, no further disease progression was generally observed.
Children with RNASEH2B mutations presented significantly later than did those with mutations in TREX1, RNASEH2C, or RNASEH2A (Mann-Whitney U test, P. Age (mo) at presentation by gene for patients with TREX1, RNASEH2A, RNASEH2B, and RNASEH2C mutations. With the Mann-Whitney U test comparing age at presentation for patients with RNASEH2B mutations with that for patients with mutations in TREX1, RNASEH2C, and RNASEH2A, P18 years (the oldest known patient is aged 25 years). Age at death (in years) of patients with TREX1, RNASEH2A, RNASEH2B, and RNASEH2C mutations; χ 2 test comparing number of deaths among children with TREX1, RNASEH2C, and RNASEH2A mutations against number of deaths among children with RNASEH2B mutations ( P=.001).
Chilblain lesions were reported in 43% of patients and were associated with mutations in all four genes. The lesions were usually situated on the feet but sometimes also affected the hands and outer rim of the ears. Many parents reported a direct relationship with cold temperatures, so the lesions were considerably worse during winter months. Radiographic Findings Neuroimaging demonstrated intracranial calcification variably involving the basal ganglia, dentate nuclei of the cerebellum, and deep white matter.
Additionally, there were associated abnormalities of the white matter, with, in milder cases, high signal on T2-weighted MRI situated at the anterior and posterior poles of the lateral ventricles and, in more severely affected patients, a striking frontotemporal leukodystrophy with temporal cystic lesions. In those subjects presenting in the perinatal period, calcifications and white-matter disease were evident as early as the 1st d of life.
Cortical atrophy was a common feature in later scans, and a number of children demonstrated significant brain-stem and cerebellar atrophy. Thinning and, in one subject, complete absence of the corpus callosum were also observed. Laboratory Findings Where assayed, the number of white cells in the CSF was normal (5 cells/mm 3 was documented was 9 years (Kruskal-Wallis test comparing CSF WCC with age group, P.
Multiple of the Upper Limit of Normal Gene and Age at Examination Neopterin Biopterin Tetrahydrobio-Pterin Dihydrobio-Pterin WCC/mm 3 IFN-α TREX1: 11 mo 16 1.5 NA NA 33 25 IU/liter 14 mo 40 1.5 NA NA 3 NA. Discussion AGS is one of a number of disorders whose clinical features mimic the sequelae of in utero viral infection., – These conditions are important to recognize because of the associated high risk of recurrence. Our experience indicates that the possibility of a genetic disorder in subjects with AGS is sometimes unrecognized until the birth of a second affected child. In this regard, we note that no mention was made of such genetic disorders in a recent review of congenital infection.
We suggest that an absence of definitive evidence of an infectious agent in these circumstances should always raise the suspicion of AGS. A subgroup of AGS-affected patients, typically those with TREX1 mutations, presented at birth with abnormal neurology, hepatosplenomegaly, elevated liver enzymes, and thrombocytopenia, a picture highly reminiscent of congenital infection. All other children presented at variable times beyond the first few days of life, frequently after a period of apparently normal development. The majority of these later-presenting cases exhibited a severe encephalopathy with subacute onset that was characterized by extreme irritability, intermittent sterile pyrexias, a loss of skills, and a slowing of head growth.
This encephalopathic phase usually lasted several months. Interestingly, the opinion of most pediatricians involved in the care of these children was that there was no disease progression beyond the encephalopathic period. When death occurred, the death was usually considered not to be due to a regressive process but to be secondary to the neurological damage incurred during the initial disease episode. RNASEH2B mutations were associated with a significantly later age at presentation, at or after age 12 mo in five cases, and a lower mortality, with seven patients known to be alive beyond age 18 years with no signs of disease progression. Also of note, six affected individuals with RNASEH2B mutations demonstrated relatively preserved intellectual function; one patient has a completely normal intelligence quotient and head circumference at age 19 years, and his only feature is a spastic cerebral palsy with associated intracranial calcification.
The observation of changes on brain imaging at birth indicates an in utero onset of the disease process, in keeping with the recording of prenatal raised levels of IFN-α. In contrast, our data also highlight the onset of AGS after many months of normal development, raising the possibility that the condition might occur in considerably older individuals too. The stimulus for the disease onset is unknown, and why the disease tends to “burn out” after several months is also not understood. The cardinal features of AGS on brain imaging were intracranial calcification, a leukodystrophy, and cerebral atrophy.
The distribution and extent of the calcification was variable and, in some cases, was observed in a periventricular distribution highly suggestive of congenital infection. Affected sibling pairs have been described as discordant for the presence of intracranial calcification, so this feature should not be considered a prerequisite for the diagnosis of AGS. Additionally, in several of our patients, the intracranial calcification became evident only over a period of months.
Of importance, intracranial calcification is not always recognized on MRI, the initial imaging modality employed in most medical facilities. Consequently, AGS should be considered in the differential diagnosis of an unexplained leukoencephalopathy, and CT scanning is warranted in cases conforming to the clinical scenarios we have outlined above. Of further note, some patients demonstrated marked frontotemporal white-matter involvement with cyst formation, so that Alexander disease (MIM ) and vanishing white-matter disease (MIM ) were considered and tested for. We identified 99 families with biallelic mutations in one of the four genes known to cause AGS. Of these families, 78.8% had mutations in either TREX1 or RNASEH2B. Moreover, all the patients with RNASEH2B mutations harbored at least one mutation in exon 2, 6, or 7. Thus, an initial screen of TREX1 plus these three RNASEH2B exons, together with an analysis for the recurrent c.205C→T (R69W) RNASEH2C mutation in Pakistani patients, is likely to identify 90% of all AGS-affected patients with mutations in one of these four genes.
In four families, we were able to identify only a single RNASEH2A (one family) or RNASEH2B (three families) mutation, and one further child carried two putative RNASEH2A mutations on the same allele; these cases are under further study. In addition, we studied 22 families in whom no AGS1–4 mutations could be identified. Given the high detection rate of biallelic mutations in AGS1–4, this finding strongly suggests further genetic heterogeneity. Finally, only a single patient was observed with a de novo heterozygous TREX1 mutation, indicating that such cases are unusual.
The majority of TREX1 mutations are null alleles that are predicted to produce truncated protein, which might be inactive or disrupt protein complexes in the cell. Our recent description of heterozygous TREX1 mutations as a cause of dominant AGS suggests that some mutations may have a dominant negative effect.
Additionally, multiple patients are homozygous or compound heterozygous for a recurrent amino acid substitution, R114H, known to be involved in stabilization of the TREX1 dimer and that appears to abrogate TREX1 activity. In contrast to TREX1, almost all mutations identified in RNASEH2A, RNASEH2B, and RNASEH2C are missense changes, which suggests a hypomorphic effect rather than a complete loss of RNASEH2 enzyme function. Indeed, this mutational spectrum raises the possibility that biallelic null alleles in any of these three genes might be lethal during embryonic development.
Minimum diagnostic criteria for AGS are difficult to define. We have already highlighted above the absence of intracranial calcification in some cases of AGS. A CSF lymphocytosis was originally described as a primary diagnostic feature of the disease and was posited to differentiate AGS from pseudo-TORCH syndrome. However, it is well recognized that the level of white cells and IFN-α in the CSF of AGS-affected patients falls to normal over the first few years of life. Moreover, in our series, a normal CSF WCC was documented in the presence of elevated CSF IFN-α titers on 15 occasions. Recently described a possible variant of AGS associated with high levels of CSF pterins.
A number of mutation-positive AGS subjects in our series demonstrated a similar pterin profile, which should now be considered a marker of AGS. Whether the cases described by Blau et al. Have AGS or a separate condition remains to be determined.
The chilblain lesions seen in 43% of AGS cases provide a useful clinical marker of the disease and indicate an immune pathology. The observation of a small number of AGS-affected children with autoantibodies, hypothyroidism, and IDDM also suggests immune dysfunction. We recently described heterozygous TREX1 mutations in an autosomal dominant cutaneous form of systemic lupus erythematosus (SLE MIM ) called “familial chilblain lupus” (MIM ). The precise functions of TREX1 and the RNASEH2 complex are unknown. We predict that these nucleases are involved in removing nucleic acid species produced during apoptosis and that a failure of this process results in activation of the innate immune system., This hypothesis would explain the phenotypic overlap of AGS with congenital infection and some aspects of SLE, – where an IFN-α–mediated innate immune response is triggered by viral and host nucleic acids, respectively.