Identification of novel mutations in the HbF repressor gene <i>BCL11A</i> in patients with autism and intelligence disabilities
Authors: Tao Cai, Xiang Chen, Jinchen Li, Bingwu Xiang, Liu Yang, Yidian Liu, Qiuli Chen, Zhouwen He, Kevin Sun, P. Paul Liu
Journal: American Journal of Hematology (2017)

Genome-wide association studies demonstrated that the level of fetal hemoglobin (HbF) is associated with the BCL11A-containing region at chromosome 2p15-p16.1.1 Increased HbF levels were recently observed in multiple unrelated patients with various lengths of microdeletions in the region (eg, from 2.5-Mb deletion in the upstream region to 3.5-Mb deletion in the distal downstream region of BCL11A),2 suggesting contiguous gene effects on HbF levels and neurodevelopment of the affected individuals. However, it is not clear whether an elevation of HbF levels would result from BCL11A point mutations. Here, we present clinical evidence that a loss-of-function (LoF) mutation due to a single nucleotide substitution of the gene, but not deletions, caused a significant elevation of HbF level as well as several other developmental abnormalities. In genetic screens of 60 trios with autism spectrum disorders (ASD) and intelligence disabilities (ID) by whole exome sequencing (WES) (Supporting Information Methods),3 we identified a nonsense mutation (c.644C>G; p.Ser215*) in exon 4 of BCL11A (GenBank acc. no., NM_022893) from a 3.5-year-old boy with ID and microcephaly (Figure 1A). The pathogenic cause of the disorder was assumed to be a de novo heterozygous mutation in a single gene, and confirmed by Sanger sequencing (Figure 1B). The parents were phenotypically and genotypically normal. In an additional screen of 256 trios with ASD by targeted sequencing of the BCL11A-containing exomes, we identified a novel heterozygous frameshift mutation (c.1826delC; p.P609fs) in a 6-year-old boy and a heterozygous missense mutation (c.56C>T; p.Pro19Leu) in a 5-year-old boy (Supporting Information Table S1). Analyses of BCL11A mutations and HbF levels in erythroid cells. A, The 3.5-year-old proband with ID and his family tree. B, Bidirectional Sanger sequencing confirmed that the variant c.644C>G is detected in the proband, but not his parents. C, Schematic representation of two BCL11A protein isoforms (L and XL) with zinc-finger motifs (ZnF) in vertical boxes and reported mutations labeled. An additional LoF mutation at Pro609 in a patient with ID and a missense mutation at Pro19 in a patient with ASD are detected by WES. Four different missense variants (Supporting Information Table S1) found in five phenotypically normal individuals are also depicted. The zinc-finger motifs are predicted by SMART program (http://smart.embl-heidelberg.de/). D,E, HPCL analysis shows that HbF level in peripheral blood is elevated in the proband with p.Ser215* (D), but not in the age- and gender-matched control (E) These three variants were mapped to different zinc-finger motif-flanking regions of the encoded protein (Figure 1C), and all predicted to be disease-causing by MutationTaster and several commonly used algorithms. The stop-gain mutation p.Ser215* was predicted to produce a truncated protein (missing 621 amino acids), thereby disrupting the protein's function. The indel mutation c.1826delC was also predicted to be a LoF mutation because the last 227 amino acids were not translated due to a frameshift at p.Pro609 (Supporting Information Table S1). Furthermore, none of these variants was found in ExAC database or the inhouse database of Chinese Han Exome Sequencing (ChES). Notably, not even a single LoF allele of BCL11A could be found in the ChES database, given the fact that BCL11A is extremely conserved evolutionarily. However, we found four private missense mutations of BCL11A (p.Asp42Asn, p.Leu186Ser, p.Leu360Pro, and p.Thr400Met) in the ChES database (Figure 1C and Supporting Information Table S1), in which the p.Asp42Asn was paternally inherited. Although these four variants were predicted to be disease causing, clinical assessments confirmed that all the carriers were phenotypically normal. Detailed clinical analyses revealed that the proband carrying the LoF mutation (p.Ser215*) had multiple conditions, including severe ID (total DQ score was 20, Gesell Development Scale), microcephaly (head circumference: 43.5 cm, −5 SDS), significant language delay (apraxia of speech and oral-motor dyspraxia), strabismus, depressed nasal bridge, high palate arch, thin (body weight: 12 kg, −1.3 SDS), and short stature (height: 87 cm, −3.5 SDS). MRI analysis showed a mild brain hypoplasia at 1.5-year-old (Supporting Information Figure S1A-C), and appeared to be normal at 3.5-year-old (Supporting Information Figure S1D-F). A lower spine scoliosis in the proband (Supporting Information Figure S2A,B) was not reported in previous cases with BCL11A mutations (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php). BCL11A is abundantly expressed in the developing cerebral cortex (Supporting Information Figure S3A-C). BCL11A [MIM 606557] encodes a C2H2 type zinc-finger protein that regulates transcription and is highly expressed in the adult erythroid precursor cells and lymphocytes. Notably, BCL11A mRNA expression was reduced in the expanded peripheral erythroblasts from the patient with p.Ser215* mutation by quantitative RT-PCR (Supporting Information Figure S4A-C). Elevated level of HbF in the patient was first found at 2.5 years of age (2.48%, normally in Chinese <2%, Supporting Information Table S2) via the alkali denaturation test. This finding was confirmed by HPLC analysis at 3.5 years of age (Figure 1D,E). This elevation due to a single base substitution of BCL11A is similar to the altered HbF levels in patients with various microdeletions of BCL11A-associated regions,4 suggesting that the encoded BCL11A protein plays an important role as a developmental stage-specific repressor of HbF expression.5 Additional blood examination revealed that most hematological parameters were largely normal in the patient (Supporting Information Table S2). On the other hand, HbF was not elevated in the patient with a missense mutation (c.56C>T; p.Pro19Leu), nor in the other BCL11A-mutation carriers without neuropsychological abnormalities (Supporting Information Table S1). In the study of human malignancies, BCL11A was found to be a stable subunit of the mammalian SWI/SNF chromatin remodeling complexes implicated as tumor suppressors. Mutations in BCL11A were frequently found in glioma or neuroblastoma. Given the critical role of BCL11A in B cell development, overexpression of the gene in mice seems to promote B cell oncogenesis. Recurrent involvement and deregulated expression of BCL11A were observed in multiple cases of B-cell malignancy with the translocation t(2;14)(p13;q32.3).6 Therefore, attentions should be given on whether alterations of BCL11A expression and function induce lymphocyte malignancies in the patients with mutations identified in current and previous studies. Since an adequate reduction of BCL11A in erythroid cells will increase HbF expression, various attempts, such as inactivation of BCL11A in mice, erythroid-specific expression of shRNAmiR targeting BCL11A, deletion of the Bcl11a-enhancer, and potential application of several BCL11A-targeting miRNAs and the immunomodulatory drug Pomalidomide, have been made to ameliorate the severity of sickle cell disease (SCD) and β-thalassemia. These findings provide strong supports for genetic modifications of BCL11A as potential therapies of beta-hemoglobin disorders. However, it is worth noting that the phenotypically normal individuals carrying hypomorphic BCL11A alleles did not show an elevated HbF level. Nevertheless, long-term safety of the interventions requires further investigations in appropriate animal models before more aggressive approaches with genetic modification of BCL11A can be applied for humans. In conclusion, our findings expanded the spectrum of BCL11A mutations and associated clinical manifestations. Specially, a persistent elevation of HbF level was confirmed to be present in the individual with a nonsense mutation of BCL11A, but not present in several other individuals with missense alleles. The finding suggests that elevated HbF level can be achieved in patients with BLC11A point mutations. Authors are indebted to the individuals and their family members who participated in this study. The authors declare no competing financial interests. The study received approval from the institutional review board of Wenzhou Medical University. T.C., X.C. and P.P.L. designed the research study, analyzed the data. T.C. wrote the paper. L.Y., J.L., B.X., W.C., Q.C., and K.S. gave a substantial contribute to the acquisition of data. All authors approved the final version. Additional Supporting Information may be found online in the supporting information tab for this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.