.2015 Feb;23(2):165-72.

doi: 10.1038/ejhg.2014.82. Epub 2014 Apr 30.

Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans

A Gulhan Ercan-Sencicek¹, Samira Jambi², Daniel Franjic³, Sayoko Nishimura¹, Mingfeng Li³, Paul El-Fishawy⁴, Thomas M Morgan⁵, Stephan J Sanders⁶, Kaya Bilguvar⁷, Mohnish Suri⁸, Michele H Johnson⁹, Abha R Gupta¹⁰, Zafer Yuksel¹¹, Shrikant Mane¹², Elena Grigorenko¹³, Marina Picciotto¹⁴, Arthur S Alberts¹⁵, Murat Gunel¹⁶, Nenad Šestan³, Matthew W State⁶

Affiliations

¹ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven, CT, USA.
² Al Hada Armed Hospitals, Taif, Saudi Arabia.
³ Department of Neurobiology and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA.
⁴ Child Study Center, Yale University School of Medicine, New Haven, CT, USA.
⁵ Department of Pediatrics, Vanderbilt University, Nashville, TN, USA.
⁶ Department of Psychiatry, University of California, School of Medicine, San Francisco, CA, USA.
⁷ 1] Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven, CT, USA [2] Yale Center for Mendelian Genomics, New Haven, CT, USA.
⁸ Nottingham Clinical Genetics Service, Nottingham University Hospitals NHS, Trust City Hospital Campus, Nottingham, UK.
⁹ Departments of Diagnostic Radiology, Surgery and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA.
¹⁰ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA.
¹¹ Department of Medical Genetics, Osmangazi University, School of Medicine, Eskisehir, Turkey.
¹² 1] Yale Center for Mendelian Genomics, New Haven, CT, USA [2] Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
¹³ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Moscow State University for Psychology and Education, Moscow, Russia.
¹⁴ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Department of Neurobiology and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA [3] Departments of Psychiatry, Pharmocology Yale University School of Medicine, New Haven, CT, USA.
¹⁵ Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, MI, USA.
¹⁶ Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven, CT, USA.

PMID:24781755
PMCID: PMC4297910
DOI: 10.1038/ejhg.2014.82

Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans

A Gulhan Ercan-Sencicek et al. Eur J Hum Genet.2015 Feb.

.2015 Feb;23(2):165-72.

doi: 10.1038/ejhg.2014.82. Epub 2014 Apr 30.

Authors

Affiliations

¹ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven, CT, USA.
² Al Hada Armed Hospitals, Taif, Saudi Arabia.
³ Department of Neurobiology and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA.
⁴ Child Study Center, Yale University School of Medicine, New Haven, CT, USA.
⁵ Department of Pediatrics, Vanderbilt University, Nashville, TN, USA.
⁶ Department of Psychiatry, University of California, School of Medicine, San Francisco, CA, USA.
⁷ 1] Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven, CT, USA [2] Yale Center for Mendelian Genomics, New Haven, CT, USA.
⁸ Nottingham Clinical Genetics Service, Nottingham University Hospitals NHS, Trust City Hospital Campus, Nottingham, UK.
⁹ Departments of Diagnostic Radiology, Surgery and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA.
¹⁰ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA.
¹¹ Department of Medical Genetics, Osmangazi University, School of Medicine, Eskisehir, Turkey.
¹² 1] Yale Center for Mendelian Genomics, New Haven, CT, USA [2] Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
¹³ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Moscow State University for Psychology and Education, Moscow, Russia.
¹⁴ 1] Child Study Center, Yale University School of Medicine, New Haven, CT, USA [2] Department of Neurobiology and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA [3] Departments of Psychiatry, Pharmocology Yale University School of Medicine, New Haven, CT, USA.
¹⁵ Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, MI, USA.
¹⁶ Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven, CT, USA.

PMID:24781755
PMCID: PMC4297910
DOI: 10.1038/ejhg.2014.82

Abstract

The combination of family-based linkage analysis with high-throughput sequencing is a powerful approach to identifying rare genetic variants that contribute to genetically heterogeneous syndromes. Using parametric multipoint linkage analysis and whole exome sequencing, we have identified a gene responsible for microcephaly (MCP), severe visual impairment, intellectual disability, and short stature through the mapping of a homozygous nonsense alteration in a multiply-affected consanguineous family. This gene, DIAPH1, encodes the mammalian Diaphanous-related formin (mDia1), a member of the diaphanous-related formin family of Rho effector proteins. Upon the activation of GTP-bound Rho, mDia1 generates linear actin filaments in the maintenance of polarity during adhesion, migration, and division in immune cells and neuroepithelial cells, and in driving tangential migration of cortical interneurons in the rodent. Here, we show that patients with a homozygous nonsense DIAPH1 alteration (p.Gln778*) have MCP as well as reduced height and weight. diap1 (mDia1 knockout (KO))-deficient mice have grossly normal body and brain size. However, our histological analysis of diap1 KO mouse coronal brain sections at early and postnatal stages shows unilateral ventricular enlargement, indicating that this mutant mouse shows both important similarities as well as differences with human pathology. We also found that mDia1 protein is expressed in human neuronal precursor cells during mitotic cell division and has a major impact in the regulation of spindle formation and cell division.

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Figures

**Figure 1**
The pedigree structure and radiographic features of the SAR1008 family. (a) The patients described in this study are numbered. Parents are first cousins and have three affected female (IV:4, IV:5, IV:6), two affected male (IV:7, IV:8) and one unaffected female (IV:2) children. Squares and circles indicate males and females, respectively. Affected individuals are shown as filled symbols. One pregnancy that resulted in miscarriage is shown as a triangle. A diagonal line through a symbol denotes that the individual is deceased. The individuals from whom DNAs were obtained are indicated by *. (b) Representative MRIs of individual IV:7 at 4 months of age and control subject at 3 years of age. Axial T2 and T1 at the level of the temporal lobe, T2 and T1 at the level of the chiasm, and T1 sagittal images are shown (left to right). The MRI reveals temporal pole atrophy with normal cortical thickness. The optic nerves (on) and chiasm (oc) appear normal in size (indicated by arrows). Midline sagittal T1 shows hypoplasia of the rostrum (r) and splenium (s) of the corpus callosum. The scale bars are in cm.

**Figure 2**
Identification of a nonsense homozygous sequence variant in theDIAPH1 gene in a family with MCP. (a) Panel shows the results of the parametric linkage analysis for chromosome 5q, expressed as LOD scores. The maximum theoretical LOD score for this family is 3.7 using the genotyping data from IMv1 Duo Bead array chips. The analysis is modeled under the recessive mode of inheritance, penetrance of 99%, a phenocopy rate of 0.01, and a disease allele frequency of 0.0001. (b) Domain organization of mDia1. Abbreviations: GBD, GTPase binding region; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain. Nonsense sequence variant p.Gln778* results in a 86.2 kDa truncated protein. (c) mRNA levels ofDIAPH1 gene in EBV transformed LCLs from homozygous patients (IV:4, IV:5, IV:6, IV:7), and heterozygous parents (III:1 and III:2) were analyzed using RT-PCR. Three individuals of the same ethnicity and without a sequence variant are used as control (WT). (d) Protein blot for mDia1. Lane 1 represents the control subject, lanes 2 and 3 represent the unaffected parents who carry the heterozygous p.Gln778* alteration (III:1 and III:2). Lanes 4, 5, 6, and 7 represent affected individuals with homozygous p.Gln778* alteration (IV:4, IV:5, IV:6, IV:7). The band indicates a molecular weight of ∼140 kDa. GAPDH is used as control (lower band).

**Figure 3**
Expression pattern and subcellular localization of DIAPH1 in E14.5-day-old mouse brain (this period of development is equivalent to human brain 12 PCW), fetal human brain, and mitotic human cortical neural progenitor cells. A set of two panels from a developing mouse brain (E14.5) indicates (a) PAX6 (under the white line) at VZ, and (b)*in situ* hybridization showsDiap1 expression at VZ and SVZ. (c)*In situ* hybridization of early fetal human brain (12 PCW) showsDIAPH1 expression in the VZ and SVZ. The scale bar indicates 100 μm and 600 μm, respectively. See Supplementary Figure 3 for individual panels. (d) Confocal microscopy analysis of fixed human cerebral cortical neural progenitor cells in mitosis (M phase). Cells were co-stained with anti-mDia1 (green), pericentrin (red), and DAPI for DNA (blue). The scale bar indicates 10 μM.

**Figure 4**
Cortical organization is preserved in theDiap1^−/− mouse. Image depicts deeper cortical layer marker TBR1, layer VI (green), and phalloidin staining in the coronal sections of the lateral ventricle wall in (a) Wt (*Diap1*^+/+) and (b) mutant (*Diap1*^−/−) mice at E14.5 day. Boxed areas are shown at the higher magnification. Scale bars 200 μm at lower magnification and 50 μm at higher magnification. (c–d) Nissl staining of coronal brain sections of Diap1-KO and Wt at P0. Note the lateral ventricle dilatation on the Diap1-KO mouse. Scale bar is 1 mm.

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Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans

Affiliations

Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans

Authors

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Abstract

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