Bruce Bennetts

Mutations of CDKL5 Cause a Severe Neurodevelopmental Disorder with Infantile Spasms and Mental Retardation

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused, in most classic cases, by mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). A large degree of phenotypic variation has been observed in patients with RTT, both those with and without MECP2 mutations. We describe a family consisting of a proband with a phenotype that showed considerable overlap with that of RTT, her identical twin sister with autistic disorder and mild-to-moderate intellectual disability, and a brother with profound intellectual disability and seizures. No pathogenic MECP2 mutations were found in this family, and the Xq28 region that contains the MECP2 gene was not shared by the affected siblings. Three other candidate regions were identified by microsatellite mapping, including 10.3 Mb at Xp22.31-pter between Xpter and DXS1135, 19.7 Mb at Xp22.12-p22.11 between DXS1135 and DXS1214, and 16.4 Mb at Xq21.33 between DXS1196 and DXS1191. The ARX and CDKL5 genes, both of which are located within the Xp22 region, were sequenced in the affected family members, and a deletion of nucleotide 183 of the coding sequence (c.183delT) was identified in CDKL5 in the affected family members. In a screen of 44 RTT cases, a single splice-site mutation, IVS13-1G-->A, was identified in a girl with a severe phenotype overlapping RTT. In the mouse brain, Cdkl5 expression overlaps--but is not identical to--that of Mecp2, and its expression is unaffected by the loss of Mecp2. These findings confirm CDKL5 as another locus associated with epilepsy and X-linked mental retardation. These results also suggest that mutations in CDKL5 can lead to a clinical phenotype that overlaps RTT. However, it remains to be determined whether CDKL5 mutations are more prevalent in specific clinical subgroups of RTT or in other clinical presentations.

Effects of MECP2 mutation type, location and X-inactivation in modulating Rett syndrome phenotype.

Rett syndrome (RTT) is a clinically defined disorder that describes a subset of patients with mutations in the X‐linked MECP2 gene. However, there is a high degree of variability in the clinical phenotypes produced by mutations in MECP2, even amongst classical RTT patients. In a large‐scale screening project, this variability has been examined by looking at the effects of mutation type, functional domain affected and X‐inactivation. Mutations have been identified in 60% of RTT patients in this study (25% of whom were atypical), including 23 novel mutations and polymorphisms. More mutations were found in classical patients (63%) compared to atypical patients (44%). All of the pathogenic mutations were de novo in patients for whom parent DNA was available for screening. A composite phenotype score was developed, based on the recommendations for reporting clinical features in RTT of an international collaborative group. This score proved useful for summarising phenotypic severity, but did not correlate with mutation type, domain affected or X‐inactivation, probably due to complex interactions between all three. Other correlations suggested that truncating mutations and mutations affecting the methyl‐CpG‐binding domain tend to lead to a more severe phenotype. Skewed X‐inactivation was found in a large proportion (43%) of our patients, particularly in those with truncating mutations and mutations affecting the MBD. It is therefore likely that X‐inactivation does modulate the phenotype in RTT.

Increased frequency of CCR-5 Δ32 heterozygotes among long-term non-progressors with HIV-1 infection

Background:The β-chemokine receptor CCR-5 is used as a coreceptor by macrophage-tropic strains of HIV-1 to gain entry into CD4+ cells. Objective:To determine the effect of a common 32 base-pair deletion mutation in the CCR-5 gene (CCR-5 Δ32) on progression of HIV infection to AIDS, and to assess the level of heterozygosity for this mutation in a well-defined group of long-term non-progressors (LTNP). Participants:Sixty-four HIV-1-infected LTNP (CD4+ T lymphocyte count > 500 × 106/l after 8 years) were compared with 95 individuals infected within a similar period (1983–1986) but who had rapidly progressed to AIDS and death, and with a further 120 HIV-positive individuals with CD4+ counts < 500 × 106/l. Methods:The presence of the CCR-5 Δ32 mutation was assessed using polymerase chain reaction with primers spanning the 32 base-pair deletion. CD4+ and CD8+ counts, plasma HIV-1 RNA, p24 antigen and β2-microglobulin levels in LTNP carrying the CCR-5 Δ32 mutation were compared with LTNP lacking the mutation. Results:A marked increase in the frequency of CCR-5 Δ32 heterozygosity was found among LTNP (35.9%) compared with rapid progressors (12.6%; P = 0.0005) and patients selected on the basis of a CD4+ T-cell count < 500 × 106/l (12.5%; P = 0.0004). LTNP heterozygous for CCR-5 Δ32 had a significantly higher CD8+ T-cell count than those without the mutation (1218 versus 972 × 106/l; P = 0.044). No significant correlation was observed between heterozygosity and CD4 count, viral load, p24 antigen or β2-microglobulin within the LTNP group. Conclusions:This study provides the strongest evidence to date for the importance of a single copy of the CCR-5 Δ32 mutation in long-term non-progression of HIV infection, which may involve, in part, CD8+ T lymphocytes.To determine the effect of a common 32 base-pair deletion mutation in the CCR-5gene (CCR-5 Δ32) on progression of HIV infection to AIDS, and to assess the level of heterozygosity for this mutation in a well-defined group of long-term non-progressors (LTNP).

Identification of 11 novel and common single nucleotide polymorphisms in the interleukin-7 receptor-|[alpha]| gene and their associations with multiple sclerosis

We have investigated the interleukin-7 receptor (IL-7R) α-chain gene as a positional and functional candidate gene for susceptibility to multiple sclerosis (MS), in view of its chromosomal location on 5p14–p12, a region that has shown suggestive linkage in MS genome screens, and its role in T- and B-cell proliferation and reactivity. Amplification and DNA sequencing of the IL-7Rα gene in pooled and individual samples identified 13 single nucleotide polymorphisms (SNPs), 11 of which are novel, including three in the promoter region, three in exons encoding amino-acid changes (ACC(Thr)66ATC(Ile), ATC(Ile)244ACC(Thr), ATC(Ile)336GTC(Val)), four in introns and one in the 3′ untranslated region. Four IL-7R haplotypes were identified for nine SNPs, showing linkage disequilibrium across the gene, and allowing haplotype frequency determination from just three of the nine SNPs. Genotyping of the −504 polymorphism in 101 MS and 90 controls showed a suggestive (P=0.1) association of the T allele with MS; however, this was not supported by transmission disequilibrium testing in 186 MS trio families (P=0.8). There were trends towards an increase of the GTG+ haplotype (odds ratio=1.45), and under-representation of the TTA+ haplotype (OR=0.65) in DRB1*1501-positive MS cases, suggesting that larger sample sizes and comparison in more defined MS patient groups may support an association with the IL-7R gene. These polymorphisms would also be useful for studying genetic associations with other immunologic diseases.

The CCR5 Deletion Mutation Fails to Protect Against Multiple Sclerosis

Recent advances in the understanding and identification of chemokines and their receptors have provided evidence for their consideration as candidate loci with respect to genetic susceptibility/resistance to MS. Increased levels of the chemokine, macrophage inflammatory protein (MIP)-1 alpha, have been demonstrated in the cerebrospinal fluid of both patients with MS and mice with EAE, and anti-MIP-1 alpha antibodies have been shown to prevent EAE. Recently, a common deletion mutation in the gene for the major receptor for MIP-1 alpha, chemokine receptor 5 (CCR5) has been described. Homozygotes for the mutation fail to express this receptor. Moreover, homozygotes are highly protected against HIV infection this has potential implications for the cell entry of infectious agents in other multifactorial disease where a viral component may be involved. In view of these aspects, a group of 120 unrelated Australian relapsing remitting MS and 168 unrelated control subjects were screened for the CCR5 delta 32 mutation. There was no significant difference in the allele frequency of CCR5 delta 32 gene between the MS patients (0.1125) and the control population (0.0921). The presence of two CCR5 delta 32 homozygotes in the MS patients indicates that the absence of CCR5 is not protective against MS. These data suggest that CCR5 is not an essential component in MS expression, though this may be due to redundancy in the chemokine system where different chemokine receptors may substitute for CCR5 when it is absent.

FBN1, TGFBR1, and the Marfan-craniosynostosis/mental retardation disorders revisited.

The recent identification of TGFBR2 mutations in Marfan syndrome II (MFSII) [Mizuguchi et al. (2004); Nat Genet 36:855–860] and of TGFBR1 and TGFBR2 mutations in Loeys–Dietz aortic aneurysm syndrome (LDS) [Loeys et al. (2005); Nat Genet 37:275–281] [OMIM 609192] has provided direct evidence of abnormal signaling in transforming growth factors β (TGF‐β) in the pathogenesis of Marfan syndrome (MFS). In light of this, we describe the phenotypes and genotypes of five individuals. Patient 1 had MFS and abnormal cranial dura. Patient 2 had severe early onset MFS and an abnormal skull. Patients 3 and 4 had probable Furlong syndrome (FS). Patient 5 had marfanoid (MD) features, mental retardation (MR), and a deletion of chromosome 15q21.1q21.3. All patients had a condition within the MFS, MD‐craniosynostosis (CS) or MD‐MR spectrum. The names of these entities may become redundant, and instead, come to be considered within the spectrum of TGF‐β signaling pathway disorders. Two recurrent heterozygous FBN1 mutations were found in Patients 1 and 2, and an identical novel heterozygous de novo TGFBR1 mutation was found in Patients 3 and 4, in whom altered fibrillin‐1 processing was demonstrated previously [Milewicz et al. (2000); Am J Hum Genet 67:279]. A heterozygous FBN1 deletion was found in Patient 5. These findings support the notion that perturbation of extracellular matrix homeostasis and/or remodeling caused by abnormal TGF‐β signaling is the core pathogenetic mechanism in MFS and related entities including the MD‐CS syndromes.

Guidelines for the genetic diagnosis of hereditary recurrent fevers

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinflammatory diseases characterised by recurrent bouts of fever and serosal inflammation that are caused by pathogenic variants in genes important for the regulation of innate immunity. Discovery of the molecular defects responsible for these diseases has initiated genetic diagnostics in many countries around the world, including the Middle East, Europe, USA, Japan and Australia. However, diverse testing methods and reporting practices are employed and there is a clear need for consensus guidelines for HRF genetic testing. Draft guidelines were prepared based on current practice deduced from previous HRF external quality assurance schemes and data from the literature. The draft document was disseminated through the European Molecular Genetics Quality Network for broader consultation and amendment. A workshop was held in Bruges (Belgium) on 18 and 19 September 2011 to ratify the draft and obtain a final consensus document. An agreed set of best practice guidelines was proposed for genetic diagnostic testing of HRFs, for reporting the genetic results and for defining their clinical significance.

A genome screen for linkage in Australian sibling-pairs with multiple sclerosis

The role of genetic factors in determining susceptibility to multiple sclerosis is well established but, despite the global distribution of the disease, systematic efforts to locate susceptibility genes have concentrated exclusively on populations from the Northern Hemisphere. We performed a genome wide screen of linkage in the Australian population using a panel of 397 microsatellite markers in 54 affected sibling-pairs. Multipoint linkage analysis revealed four regions of suggestive linkage (on chromosomes 2p13, 4q26-28, 6q26 and Xp11) and 18 additional regions of potential linkage (at 1q43-44, 3q13-24, 4q24, 4q31-34, 5q11-13, 6q27, 7q33-35, 8p23-21, 9q21, 13q31-32, 16p13, 16p11, 16q23-24, 17p13, 18p11, 20p12-11, Xp21-11 and Xq23-28). Our results contribute to the available data adding new provisional regions of linkage as well as increasing support for areas previously implicated in genetic susceptibility to multiple sclerosis.

Ectopia lentis phenotypes and the FBN1 gene

Mutations of the fibrillin‐1 (FBN1) gene on chromosome 15 have been described in patients with classical Marfan syndrome (MFS), neonatal MFS, the “MASS” phenotype, autosomal dominant ascending aortic aneurysms, autosomal dominant ectopia lentis (EL), Marfanoid skeletal features [Milewicz et al., 1995: J Clin Invest 95:2373–2378], familial arachnodactyly, Shprintzen–Goldberg syndrome [Hayward et al., 1994: Mol Cell Probes 8:325–327; Furthmayr and Francke, 1997: Semin Thorac Cardiovasc Surg 9:191–205], and severe progressive kyphoscoliosis [Adès et al., 2002: Am J Med Genet 109:261–270]. We report the use of denaturing high performance liquid chromatography (DHPLC) to facilitate the characterization of a previously elusive FBN1 mutation in the large autosomal dominant EL kindred described by Edwards et al. [1994: Am J Med Genet 53:65–71]. This isolated EL kindred remains the largest for which detailed clinical data is available. Nine years on, we present an update of the clinical status of the family. We report a recurrent FBN1 mutation, R240C, in the kindred. This mutation has been reported three times before, once in a family with classic MFS [Loeys et al., 2001: Arch Intern Med 161:2447–2454], once in one member of a multi‐generation EL kindred, [Körkkö et al., 2002: J Med Genet 39:34–41], and once in an adult from a familial EL kindred who had EL, and involvement of the integument, without cardiovascular involvement [Comeglio et al., 2002: Br J Ophthalmol 86:1359–1362]. This is the second report of the R240C mutation in association with isolated EL, and supports the existing evidence that the R240C mutation can result in two quite distinct, yet related, phenotypes. It also raises the possibility that R240C may prove to be a relative mutational “hot‐spot” for isolated EL. We review the current literature regarding EL (isolated and other) and FBN1 mutations.

The clinical phenotype of mosaicism for genome-wide paternal uniparental disomy: Two new reports†

Recently, mosaicism for genome‐wide paternal uniparental disomy (patUPD), attributed to androgenetic/biparental mosaicism, has been shown to underlie placental mesenchymal dysplasia (PMD), a distinctive cystic placental phenotype. Manifestations of Beckwith–Wiedemann syndrome (BWS) have been observed in approximately one‐third of fetuses or liveborn infants from pregnancies complicated by PMD. There are very few reports describing liveborn individuals with proven mosaicism for genome‐wide patUPD in somatic tissues. We report two further children with complex phenotypes including some findings of BWS, congenital hyperinsulinemic hypoglycemia, prolonged feeding difficulty and failure to thrive in infancy. The first developed short stature, bilateral pheochromocytomas and progressive arterial stenoses, and the second had congenital adrenal cysts, and later developed hepatoblastoma and patchy hyperpigmentation. Leukocyte DNA methylation studies of KCNQ1OT1/LIT1 and H19 loci (11p15.5) showed almost complete loss of maternal methylation (LOM) in patient 1 and partial LOM in patient 2. Microsatellite marker panels showed whole chromosome 11 patUPD. SNP array studies in both were consistent with mosaic genome‐wide patUPD in leukocytes, while fibroblast DNA in Patient 1 showed biparental inheritance. This report further illustrates the clinical consequences of mosaicism for genome‐wide patUPD, which results in complex and variable phenotypes. Studies for genome‐wide UPD should be considered in individuals with atypical UPD phenotypes.