Karen M. Lower

Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy

Mental retardation and epilepsy often occur together. They are both heterogeneous conditions with acquired and genetic causes. Where causes are primarily genetic, major advances have been made in unraveling their molecular basis. The human X chromosome alone is estimated to harbor more than 100 genes that, when mutated, cause mental retardation. At least eight autosomal genes involved in idiopathic epilepsy have been identified, and many more have been implicated in conditions where epilepsy is a feature. We have identified mutations in an X chromosome–linked, Aristaless-related, homeobox gene (ARX), in nine families with mental retardation (syndromic and nonspecific), various forms of epilepsy, including infantile spasms and myoclonic seizures, and dystonia. Two recurrent mutations, present in seven families, result in expansion of polyalanine tracts of the ARX protein. These probably cause protein aggregation, similar to other polyalanine and polyglutamine disorders. In addition, we have identified a missense mutation within the ARX homeodomain and a truncation mutation. Thus, it would seem that mutation of ARX is a major contributor to X-linked mental retardation and epilepsy.

ATR-X Syndrome Protein Targets Tandem Repeats and Influences Allele-Specific Expression in a Size-Dependent Manner

ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.

Mutations in PHF6 are associated with Borjeson-Forssman-Lehmann syndrome

Börjeson–Forssman–Lehmann syndrome (BFLS; OMIM 301900) is characterized by moderate to severe mental retardation, epilepsy, hypogonadism, hypometabolism, obesity with marked gynecomastia, swelling of subcutaneous tissue of the face, narrow palpebral fissure and large but not deformed ears. Previously, the gene associated with BFLS was localized to 17 Mb in Xq26–q27 (refs 2–4). We have reduced this interval to roughly 9 Mb containing more than 62 genes. Among these, a novel, widely expressed zinc-finger (plant homeodomain (PHD)-like finger) gene (PHF6) had eight different missense and truncation mutations in seven familial and two sporadic cases of BFLS. Transient transfection studies with PHF6 tagged with green fluorescent protein (GFP) showed diffuse nuclear staining with prominent nucleolar accumulation. Such localization, and the presence of two PHD-like zinc fingers, is suggestive of a role for PHF6 in transcription.

Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition

It is well established that all of the cis-acting sequences required for fully regulated human α-globin expression are contained within a region of ≈120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the α-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the α-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of α-globin genes, affects the pattern of NME4 expression by altering the competition for the shared α-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.

The role of the polycomb complex in silencing α-globin gene expression in nonerythroid cells

Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human alpha- and beta-globin genes are silenced by fundamentally different mechanisms. The alpha-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the beta-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the alpha-cluster by sequences within the CpG islands associated with their promoters; the beta-globin promoters do not lie within such islands. Chromatin associated with the alpha-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The alpha- and beta-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.

The clinical picture of the Börjeson–Forssman–Lehmann syndrome in males and heterozygous females with PHF6 mutations

The usual description of the Börjeson–Forssman–Lehmann syndrome (BFLS) is that of a rare, X‐linked, partially dominant condition with severe intellectual disability, epilepsy, microcephaly, coarse facial features, long ears, short stature, obesity, gynecomastia, tapering fingers, and shortened toes. Recently, mutations have been identified in the PHF6 gene in nine families with this syndrome. The clinical history and physical findings in the affected males reveal that the phenotype is milder and more variable than previously described and evolves with age. Generally, in the first year, the babies are floppy, with failure to thrive, big ears, and small external genitalia. As schoolboys, the picture is one of learning problems, moderate short stature, with emerging truncal obesity and gynecomastia. Head circumferences are usually normal, and macrocephaly may be seen. Big ears and small genitalia remain. The toes are short and fingers tapered and malleable. In late adolescence and adult life, the classically described heavy facial appearance emerges. Some heterozygous females show milder clinical features such as tapering fingers and shortened toes. Twenty percent have significant learning problems, and 95% have skewed X inactivation. We conclude that this syndrome may be underdiagnosed in males in their early years and missed altogether in isolated heterozygous females.

Mutations in Krüppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression

In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.

Mutation screening in Börjeson-Forssman-Lehmann syndrome: identification of a novel de novo PHF6 mutation in a female patient

Background: Börjeson-Forssman-Lehmann syndrome (BFLS; MIM 301900) is an infrequently described X linked disorder caused by mutations in PHF6, a novel zinc finger gene of unknown function. Objective: To present the results of mutation screening in individuals referred for PHF6 testing and discuss the value of prior X-inactivation testing in the mothers of these individuals. Results: 25 unrelated individuals were screened (24 male, one female). Five PHF6 mutations were detected, two of which (c.940A→G and c.27_28insA) were novel. One of these new mutations, c.27_28insA, was identified in a female BFLS patient. This was shown to be a de novo mutation arising on the paternal chromosome. This is the first report of a clinically diagnosed BFLS female with a confirmed PHF6 mutation. In addition, the X-inactivation status of the mothers of 19 males with suggested clinical diagnosis of BFLS was determined. Skewed (⩾70%) X-inactivation was present in five mothers, three of whom had sons in whom a PHF6 mutation was detected. The mutation positive female also showed skewing. Conclusions: The results indicate that the success of PHF6 screening in males suspected of having BFLS is markedly increased if there is a positive family history and/or skewed X-inactivation is found in the mother.

Novel PHF6 mutation p.D333del causes Börjeson-Forssman-Lehmann syndrome

The X linked mental retardation group XLMR comprises at least 160 syndromic and non-syndromic disorders of the human X chromosome as summarised by the online catalogue of Mendelian inheritance in man (OMIM; accession date July 2002; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM). For 113 of these, the gene locus has been mapped, although the gene itself may not have been identified yet. Borjeson-Forssman-Lehmann syndrome (BFLS)1 belongs to the syndromic forms of XLMR disorders.2 BFLS is best described as a mental deficiency-endocrine disorder. This relatively rare disorder was described for the first time in 1962.1 It is characterised by moderate to severe mental retardation, excessive facial fat, large ears, marked obesity, hypogonadism, and gynaecomastia. A detailed pathoanatomical follow up of the patient from the first publication of the syndrome completed the thorough description of the phenotype.3 Although BFLS is an X chromosomal recessively inherited disorder, it may also be seen in women,2,4 perhaps as a consequence of X inactivation skewing, as has been proven by methylation specific PCR.5 Subjects with BFLS may show intra- and interfamilial phenotypic variability.6 There are other autosomal and X linked disorders with similar symptoms, making the accurate diagnosis of BFLS more difficult. The differential diagnosis of obesity related syndromes includes syndromes like Prader-Willi, Bardet-Biedl, or Wilson-Turner syndromes. However, obesity as a cardinal symptom is not necessarily linked to these loci, as was proven by a linkage analysis of obese sibs.7 Therefore, a thorough analysis of the phenotype is essential. Some features, which are quite frequent in BFLS and allow for the discrimination of this disorder from other mental retardation/obesity related syndromes are: hypogonadism, long, large ears, prominent supraorbital ridge, long philtrum, protruding lips, kyphosis, coarse face, and narrow palpebral fissures, which have been described also as lid ptosis.8 BFLS has been …

Partial Androgen Insensitivity Syndrome and t(X;5): Are There Upstream Regulatory Elements of the Androgen Receptor Gene?

Background/Aims: Two half-brothers with similar malformed genitals, who both inherited a maternally derived t(X;5)(q13;p15) translocation, have a phenotype consistent with partial androgen sensitivity syndrome. The aim was to identify the gene disrupted by the X chromosome breakpoint. Methods: The breakpoint was localized using fluorescence in situ hybridization to metaphase spreads of the translocation. Results: The breakpoint on the X chromosome of the X;5 translocation was localized to a 30-kb region. This region does not contain any identified genes or transcripts. However, the breakpoint is approximately 134 kb from the 5′ end of the androgen receptor (AR) gene. Conclusions: Genetic defects of the AR gene are collectively called androgen insensitivity syndrome and include a range of phenotypes from normal males, often with associated sterility, to XY females. The phenotype seen in the males with the t(X;5) is consistent with this syndrome. The analysis of the chromosomal abnormality suggests that this translocation may remove one or more upstream regulatory elements of the AR gene that are essential for its normal expression and its role in typical external masculinization.