Richard J. Gibbons

A newly defined X linked mental retardation syndrome associated with alpha thalassaemia.

In 1981 three northern European families were described in which a severely mentally retarded son also had haemoglobin H (Hb H) disease. These findings were of interest because Hb H disease, a relatively severe manifestation of a thalassaemia, is rare in northern Europeans although it is frequently seen in Mediterranean and Oriental racial groups in which it is not known to be associated with an increased frequency of mental retardation. Furthermore, whereas the common forms of Hb H disease are always inherited in a mendelian fashion, in these northern European families this appeared not to be so. Hb H disease occurs when a greater than 50% reduction in synthesis of the a globin chains of adult haemoglobin (Hb A, a212) results in the accumulation of excess 1 globin chains which form 14 tetramers (Hb H). The common mendelian forms of HbH disease result from mutations of both allelic a globin complexes, most commonly owing to deletions or less frequently to small rearrangements or point mutations. The a globin complex is located close to the telomere of the short arm of chromosome 16, within band 16pl3.3. By 1990, a total of 13 subjects with a thalassaemia and mental retardation (ATR) had been identified and two distinct syndromes were delineated.34 Eight patients had large (1 to 2 megabases) deletions of the tip of chromosome 16p; the clinical features of this so called ATR-16 syndrome were rather variable, in

Brachydactyly Type B: Linkage to Chromosome 9q22 and Evidence for Genetic Heterogeneity

Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and is characterized by hypoplasia or absence of the terminal portions of the index to little fingers, usually with absence of the nails. The thumbs may be of normal length but are often flattened and occasionally are bifid. The feet are similarly but less severely affected. We have performed a genomewide linkage analysis of three families with BDB, two English and one Portugese. The two English families show linkage to the same region on chromosome 9 (combined multipoint maximum LOD score 8.69 with marker D9S257). The 16-cM disease interval is defined by recombinations with markers D9S1680 and D9S1786. These two families share an identical disease haplotype over 18 markers, inclusive of D9S278-D9S280. This provides strong evidence that the English families have the same ancestral mutation, which reduces the disease interval to <12.7 cM between markers D9S257 and D9S1851 in chromosome band 9q22. In the Portuguese family, we excluded linkage to this region, a result indicating that BDB is genetically heterogeneous. Reflecting this, there were atypical clinical features in this family, with shortening of the thumbs and absence or hypoplasia of the nails of the thumb and hallux. These results enable a refined classification of BDB and identify a novel locus for digit morphogenesis in 9q22.

X linked alpha thalassaemia/mental retardation: spectrum of clinical features in three related males.

We describe three males (two brothers and a cousin) who have the X linked alpha thalassaemia/mental retardation (ATR-X) syndrome. The diagnosis, originally suspected in the brothers because of similarity in dysmorphic features to previous cases, was confirmed haematologically in the surviving brother. The cousin has less typical dysmorphism and a virtually normal routine blood count, but haemoglobin H inclusions were found in his red blood cells showing that he has the same condition. This report expands the clinical phenotype of the ATR-X syndrome and emphasises that a normal blood count does not exclude the diagnosis.

The non-deletion type of alpha thalassaemia/mental retardation: a recognisable dysmorphic syndrome with X linked inheritance.

The association of a thalassaemia and mental retardation (ATR), originally described in 1981,1 was further analysed in two papers published last year. Of 13 subjects ascertained because of their haematological abnormality, eight had deletions involving the tip of chromosome 16p, where the a globin genes lie (deletion cases)2; in the remaining five, the a globin genes appeared intact (nondeletion cases). Whereas the clinical features of the deletion cases were rather variable, the non-deletion cases showed a strikingly uniform phenotype comprising severe mental handicap, characteristic dysmorphic facies, genital abnormalities, and an unusual, mild form of haemoglobin H (Hb H) disease (a manifestation ofa thalassaemia). It was proposed that the nondeletion cases represented a distinct syndrome that probably mapped to the X chromosome3; however, in the absence of pedigrees containing multiple affected cases, the evidence for this was circumstantial. Two recent papers in this Journal4 s lend support to the conclusions of Wilkie et al3 and extend the delineation of the nondeletion ATR syndrome. Harvey et al4 described a severely retarded 21 year old male with similar haematological and dysmorphic features to the previous cases and a normal a globin genotype. A male sib who was severely retarded and said to have had a similar physical appearance had died some years previously. As pointed out by the authors, this was the first description of two affected males in a sibship and is compatible with X linked inheritance. Nearly simultaneous with the publication of Wilkie et al,3 Porteous and Burn described a 6 year old boy with an unknown retardation syndrome comprising dysmorphic facies, microcephaly, hypotonia, and small genitalia; his dead maternal uncle had shown similar clinical features, leading the authors to propose that this syndrome might be X linked. The striking similarity of their case to the non-deletion ATR syndrome prompted further haematological and molecular evaluation of the proband at the age of 7 years 3 months. The results were: haemoglobin (Hb) 10.3 g/dl, red cell count 4.55 x 1012/1, mean cell volume 73 fl, mean cell haemoglobin 23 pg, Hb electrophoresis, 2.7% Hb H; 14% of red cells contained Hb H inclusions after incubation with 1% brilliant cresyl blue, and the a globin genotype was aa/aa. These results prove that this boy has the non-deletion ATR syndrome; his uncle had been anaemic throughout life and had been treated with iron supplements, so it seems likely that he had the same condition. The boys parents had normal haematological indices. Porteous and Burn suggested that their case resembled two brothers previously thought to have an atypical form of the Coffin-Lowry syndrome and illustrated in Smiths recognizable patterns ofhuman malformation.6 Haematological evaluation of the surviving brother shows that he too has non-deletion ATR; a male cousin (his mothers sisters son), also mentally retarded, has the same condition. A fuller description of this family will appear in the next issue of the Journal. This recent work lends weight to the conclusions of Wilkie et aP in two respects. First, the non-deletion ATR syndrome shows a pattem of dysmorphic facial features and associated clinical abnormalities that is sufficiently characteristic to identify potential new cases; the diagnosis is then best confirmed by showing the presence ofHb H inclusions in the red cells. Second, the absence of male-tomale transmission in the three new pedigrees (comprising seven affected cases, all male) greatly strengthens the evidence that the non-deletion ATR syndrome is X linked. Accordingly, we propose that this syndrome3 is henceforth termed X linked a thalassaemia/mental retardation (ATR-X), to distinguish it from the separate syndrome associated with deletion of the a globin complex on chromosome 16 (ATR-16).

Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single Stranded Oligonucleotide Donors

The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4–7 fold higher than starting cell numbers within 9–12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.