Miroslava Hancarova

BCL11A deletions result in fetal hemoglobin persistence and neurodevelopmental alterations

A transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA) normally occurs within a few months after birth. Increased production of HbF after this period of infancy ameliorates clinical symptoms of the major disorders of adult β-hemoglobin: β-thalassemia and sickle cell disease. The transcription factor BCL11A silences HbF and has been an attractive therapeutic target for increasing HbF levels; however, it is not clear to what extent BCL11A inhibits HbF production or mediates other developmental functions in humans. Here, we identified and characterized 3 patients with rare microdeletions of 2p15-p16.1 who presented with an autism spectrum disorder and developmental delay. Moreover, these patients all exhibited substantial persistence of HbF but otherwise retained apparently normal hematologic and immunologic function. Of the genes within 2p15-p16.1, only BCL11A was commonly deleted in all of the patients. Evaluation of gene expression data sets from developing and adult human brains revealed that BCL11A expression patterns are similar to other genes associated with neurodevelopmental disorders. Additionally, common SNPs within the second intron of BCL11A are strongly associated with schizophrenia. Together, the study of these rare patients and orthogonal genetic data demonstrates that BCL11A plays a central role in silencing HbF in humans and implicates BCL11A as an important factor for neurodevelopment.

A patient with de novo 0.45 Mb deletion of 2p16.1: the role of BCL11A, PAPOLG, REL, and FLJ16341 in the 2p15-p16.1 microdeletion syndrome.

The 2p15‐p16.1 microdeletion syndrome is a novel, rare disorder characterized by developmental delay, intellectual disability, microcephaly, growth retardation, facial abnormalities, and other medical problems. We report here on an 11‐year‐old female showing clinical features consistent with the syndrome and carrying a de novo 0.45 Mb long deletion of the paternally derived 2p16.1 allele. The deleted region contains only three protein‐coding RefSeq genes, BCL11A, PAPOLG, and REL, and one long non‐coding RNA gene FLJ16341. Based on close phenotypic similarities with six reported patients showing typical clinical features of the syndrome, we propose that the critical region can be narrowed down further, and that these brain expressed genes can be considered candidates for the features seen in this microdeletion syndrome.

Cell-Free Fetal DNA in Maternal Plasma during Physiological Single Male Pregnancies: Methodology Issues and Kinetics

Objective: To analyze methodological influences and characterize the concentrations of cell-free fetal DNA (cffDNA) circulating in maternal plasma at different gestational ages in physiological pregnancies. Methods: We investigated 238 independent samples from single male-bearing pregnancies of different gestation age. In the other 50 pregnancies, the samples were collected three times during pregnancy (at all trimesters) to evaluate the kinetics of cffDNA. The manual and automated DNA extraction methods (Roche) were compared. cffDNA was amplified using real-time PCR method and Y-specific sequences SRY and DYS14. Total cell-free DNA circulating in maternal plasma was determined by the use of the GADPH sequence. Results: The elevation in the concentration of cffDNA during pregnancy with the highest value in the third trimester was observed independently on the DNA extraction method and on the Y-specific amplified sequence. The same is documented for the percentage of fetal DNA in total cell-free DNA in maternal plasma. It increases also in successive trimesters (8.3, 10.7 and 23.2%). Conclusions: We discuss methodological problems and describe statistical parameters of cffDNA concentrations in maternal plasma during pregnancy as the basic information for comparison with pregnancies having a pathological outcome.

Dominant variants in the splicing factor PUF60 cause a recognizable syndrome with intellectual disability, heart defects and short stature

Verheij syndrome, also called 8q24.3 microdeletion syndrome, is a rare condition characterized by ante- and postnatal growth retardation, microcephaly, vertebral anomalies, joint laxity/dislocation, developmental delay (DD), cardiac and renal defects and dysmorphic features. Recently, PUF60 (Poly-U Binding Splicing Factor 60 kDa), which encodes a component of the spliceosome, has been discussed as the best candidate gene for the Verheij syndrome phenotype, regarding the cardiac and short stature phenotype. To date, only one patient has been reported with a de novo variant in PUF60 that probably affects function (c.505C>T leading to p.(His169Tyr)) associated with DD, microcephaly, craniofacial and cardiac defects. Additional patients were required to confirm the pathogenesis of this association and further delineate the clinical spectrum. Here we report five patients with de novo heterozygous variants in PUF60 identified using whole exome sequencing. Variants included a splice-site variant (c.24+1G>C), a frameshift variant (p.(Ile136Thrfs*31)), two nonsense variants (p.(Arg448*) and p.(Lys301*)) and a missense change (p.(Val483Ala)). All six patients with a PUF60 variant (the five patients of the present study and the unique reported patient) have the same core facial gestalt as 8q24.3 microdeletions patients, associated with DD. Other findings include feeding difficulties (3/6), cardiac defects (5/6), short stature (5/6), joint laxity and/or dislocation (5/6), vertebral anomalies (3/6), bilateral microphthalmia and irido–retinal coloboma (1/6), bilateral optic nerve hypoplasia (2/6), renal anomalies (2/6) and branchial arch defects (2/6). These results confirm that PUF60 is a major driver for the developmental, craniofacial, skeletal and cardiac phenotypes associated with the 8q24.3 microdeletion.

HCFC1 loss-of-function mutations disrupt neuronal and neural progenitor cells of the developing brain

Both gain- and loss-of-function mutations have recently implicated HCFC1 in neurodevelopmental disorders. Here, we extend our previous HCFC1 over-expression studies by employing short hairpin RNA to reduce the expression of Hcfc1 in embryonic neural cells. We show that in contrast to over-expression, loss of Hcfc1 favoured proliferation of neural progenitor cells at the expense of differentiation and promoted axonal growth of post-mitotic neurons. To further support the involvement of HCFC1 in neurological disorders, we report two novel HCFC1 missense variants found in individuals with intellectual disability (ID). One of these variants, together with three previously reported HCFC1 missense variants of unknown pathogenicity, were functionally assessed using multiple cell-based assays. We show that three out of the four variants tested result in a partial loss of HCFC1 function. While over-expression of the wild-type HCFC1 caused reduction in HEK293T cell proliferation and axonal growth of neurons, these effects were alleviated upon over-expression of three of the four HCFC1 variants tested. One of these partial loss-of-function variants disrupted a nuclear localization sequence and the resulting protein displayed reduced ability to localize to the cell nucleus. The other two variants displayed negative effects on the expression of the HCFC1 target gene MMACHC, which is responsible for the metabolism of cobalamin, suggesting that these individuals may also be susceptible to cobalamin deficiency. Together, our work identifies plausible cellular consequences of missense HCFC1 variants and identifies likely and relevant disease mechanisms that converge on embryonic stages of brain development.

Identification of a patient with intellectual disability and de novo 3.7 Mb deletion supports the existence of a novel microdeletion syndrome in 2p14-p15

Microdeletions spanning 2p14-p15 have recently been described in two patients with developmental and speech delay and intellectual disability but no congenital malformations or severe facial dysmorphism. We report a 4-year-old boy with a de novo 3.7 Mb long deletion encompassing the region deleted in the previous cases. The patient had clinical features partly consistent with the published cases including intellectual disability, absent speech, microcephaly, long face, bulbous nasal tip and thin upper lip, but his overall clinical picture was more severe compared to the published patients. The identification of this additional patient and a detailed analysis of deletions identified in various patient cohorts and in normal individuals support the existence of a new rare microdeletion syndrome in 2p14-p15. Its critical region is in the vicinity of but clearly separate from the minimal region deleted in the well established 2p15-p16.1 microdeletion syndrome. A thorough comparison of the deletions and phenotypes indicates that multiple genes located in this region may be involved in intellectual functioning, and that some patients may show composite and more complex phenotypes due to deletions spanning both critical regions.

FOXP1-related intellectual disability syndrome: a recognisable entity

Background Mutations in forkhead box protein P1 (FOXP1) cause intellectual disability (ID) and specific language impairment (SLI), with or without autistic features (MIM: 613670). Despite multiple case reports no specific phenotype emerged so far. Methods We correlate clinical and molecular data of 25 novel and 23 previously reported patients with FOXP1 defects. We evaluated FOXP1 activity by an in vitro luciferase model and assessed protein stability in vitro by western blotting. Results Patients show ID, SLI, neuromotor delay (NMD) and recurrent facial features including a high broad forehead, bent downslanting palpebral fissures, ptosis and/or blepharophimosis and a bulbous nasal tip. Behavioural problems and autistic features are common. Brain, cardiac and urogenital malformations can be associated. More severe ID and NMD, sensorineural hearing loss and feeding difficulties are more common in patients with interstitial 3p deletions (14 patients) versus patients with monogenic FOXP1 defects (34 patients). Mutations result in impaired transcriptional repression and/or reduced protein stability. Conclusions FOXP1-related ID syndrome is a recognisable entity with a wide clinical spectrum and frequent systemic involvement. Our data will be helpful to evaluate genotype–phenotype correlations when interpreting next-generation sequencing data obtained in patients with ID and/or SLI and will guide clinical management.

A patient showing features of both SBBYSS and GPS supports the concept of a KAT6B-related disease spectrum, with mutations in mid-exon 18 possibly leading to combined phenotypes.

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) are two distinct clinically overlapping syndromes caused by de novo heterozygous truncating mutations in the KAT6B gene encoding lysine acetyltransferase 6B, a part of the histone H3 acetyltransferase complex. We describe an 8-year-old girl with a KAT6B mutation and a combined GPS/SBBYSS phenotype. The comparison of this patient with 61 previously published cases with KAT6B mutations and GPS, SBBYSS or combined GPS/SBBYSS phenotypes allowed us to separate the KAT6B mutations into four groups according to their position in the gene (reflecting nonsense mediated RNA decay and protein domains) and their clinical outcome. We suggest that mutations in mid-exon 18 corresponding to the C-terminal end of the acidic (Asp/Glu-rich) domain of KAT6B may have more variable expressivity leading to GPS, SBBYSS or combined phenotypes, in contrast to defects in other regions of the gene which contribute more specifically to either GPS or SBBYSS. Notwithstanding the clinical overlap, our cluster analysis of phenotypes of all known patients with KAT6B mutations supports the existence of two clinical entities, GPS and SBBYSS, as poles within the KAT6B-related disease spectrum. The awareness of these phenomena is important for qualified genetic counselling of patients with KAT6B mutations.

Deletions of 9q21.3 including NTRK2 are associated with severe phenotype

Conflict of interests: none. Grant sponsor: Czech Ministry of Health; Grant numbers: NT/14200, DRO UH Motol 00064203; Grant sponsor: European Commission; Grant numbers: CHERISH 223692, CZ.2.16/3.1.00/24022. Correspondence to: Miroslava Hancarova, Department of Biology and Medical Genetics, Charles University 2nd Faculty of Medicine and University Hospital Motol, Plzenska 130/221, 15000 Prague 5, Czech Republic. E-mail: miroslava.hancarova@lfmotol.cuni.cz Article first published online in Wiley Online Library (wileyonlinelibrary.com): 27 October 2014 DOI 10.1002/ajmg.a.36797 TO THE EDITOR:

Long term follow-up in a patient with a de novo microdeletion of 14q11.2 involving CHD8

We identified a de novo deletion of 14q11.2 in a Czech patient with developmental delay, mild autistic features, macrosomy, macrocephaly, orthognathic deformities, and dysmorphic facial features. The clinical follow‐up of the patient lasting 14 years documented changes in the facial dysmorphism from infancy to adolescence. The deletion affects approximately 200 kb of DNA with five protein‐coding genes and two snoRNA genes. Two of the protein‐coding genes, SUPT16H and CHD8, have been proposed as candidate genes for a new microdeletion syndrome. Our patient further supports the existence of this syndrome and extends its phenotypic spectrum, especially points to the possibility that orthognathic deformities may be associated with microdeletions of 14q11.2. CHD8 mutations have been found in patients with neurodevelopmental disorders and macrocephaly. The HNRNPC gene, repeatedly deleted in patients with developmental delay, is another candidate as its 5́ end is adjacent to the deletion, and the expression of this gene may be affected by position effect.