Jennie E. Murray

Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

Extreme growth failure is a common presentation of ligase IV deficiency

Ligase IV syndrome is a rare differential diagnosis for Nijmegen breakage syndrome owing to a shared predisposition to lympho‐reticular malignancies, significant microcephaly, and radiation hypersensitivity. Only 16 cases with mutations in LIG4 have been described to date with phenotypes varying from malignancy in developmentally normal individuals, to severe combined immunodeficiency and early mortality. Here, we report the identification of biallelic truncating LIG4 mutations in 11 patients with microcephalic primordial dwarfism presenting with restricted prenatal growth and extreme postnatal global growth failure (average OFC −10.1 s.d., height −5.1 s.d.). Subsequently, most patients developed thrombocytopenia and leucopenia later in childhood and many were found to have previously unrecognized immunodeficiency following molecular diagnosis. None have yet developed malignancy, though all patients tested had cellular radiosensitivity. A genotype–phenotype correlation was also noted with position of truncating mutations corresponding to disease severity. This work extends the phenotypic spectrum associated with LIG4 mutations, establishing that extreme growth retardation with microcephaly is a common presentation of bilallelic truncating mutations. Such growth failure is therefore sufficient to consider a diagnosis of LIG4 deficiency and early recognition of such cases is important as bone marrow failure, immunodeficiency, and sometimes malignancy are long term sequelae of this disorder.

Extended spectrum of human glucose-6-phosphatase catalytic subunit 3 deficiency: Novel genotypes and phenotypic variability in severe congenital neutropenia

OBJECTIVE To delineate the phenotypic and molecular spectrum of patients with a syndromic variant of severe congenital neutropenia (SCN) due to mutations in the gene encoding glucose-6-phosphatase catalytic subunit 3 (G6PC3). STUDY DESIGN Patients with syndromic SCN were characterized for associated malformations and referred to us for G6PC3 mutational analysis. RESULTS In a cohort of 31 patients with syndromic SCN, we identified 16 patients with G6PC3 deficiency including 11 patients with novel biallelic mutations. We show that nonhematologic features of G6PC3 deficiency are good predictive indicators for mutations in G6PC3. Additionally, we demonstrate genetic variability in this disease and define novel features such as growth hormone deficiency, genital malformations, disrupted bone remodeling, and abnormalities of the integument. G6PC3 mutations may be associated with hydronephrosis or facial dysmorphism. The risk of transition to myelodysplastic syndrome/acute myeloid leukemia may be lower than in other genetically defined SCN subgroups. CONCLUSIONS The phenotypic and molecular spectrum in G6PC3 deficiency is wider than previously appreciated. The risk of transition to myelodysplastic syndrome or acute myeloid leukemia may be lower in G6PC3 deficiency compared with other subgroups of SCN.

Mutations in the NHEJ Component XRCC4 Cause Primordial Dwarfism

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

A Novel Nonsense CDK5RAP2 Mutation in a Somali Child With Primary Microcephaly and Sensorineural Hearing Loss

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (−3 SDS or more) and mild‐to‐moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7 Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non‐Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co‐occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP‐array testing to guide disease gene identification where an autosomal recessive condition is plausible.

Growth in individuals with Majewski osteodysplastic primordial dwarfism type II caused by pericentrin mutations

Microcephalic primordial dwarfism (MPD) is a class of disorders characterized by intrauterine growth restriction (IUGR), impaired postnatal growth and microcephaly. Majewski osteodysplastic primordial dwarfism type II (MOPD II) is one of the more common conditions within this group. MOPD II is caused by truncating mutations in pericentrin (PCNT) and is inherited in an autosomal recessive manner. Detailed growth curves for length, weight, and OFC are presented here and derived from retrospective data from 26 individuals with MOPD II confirmed by molecular or functional studies. Severe pre‐ and postnatal growth failure is evident in MOPD II patients. The length, weight, and OFC at term (when corrected for gestational age) were −7.0, −3.9, and −4.6 standard deviation (SD) below the population mean and equivalent to the 50th centile of a 28–29‐, 31–32‐, and 30–31‐week neonate, respectively. While at skeletal maturity, the height, weight, and OFC were −10.3, −14.3, and −8.5 SD below the population mean and equivalent to the size of 3‐year 10‐ to 11‐month‐old, a 5‐year 2‐ to 3‐month‐old, and 5‐ to 6‐month‐old, respectively. During childhood, MOPD II patients grow with slowed, but fairly constant growth velocities and show no evidence of any pubertal growth spurt. Treatment with human growth hormone (n = 11) did not lead to any significant improvement in final stature. The growth charts presented here will be of assistance with diagnosis and management of MOPD II, and should have particular utility in nutritional management of MOPD II during infancy.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

A syndromic form of Pierre Robin sequence is caused by 5q23 deletions encompassing FBN2 and PHAX.

Pierre Robin sequence (PRS) is an aetiologically distinct subgroup of cleft palate. We aimed to define the critical genomic interval from five different 5q22-5q31 deletions associated with PRS or PRS-associated features and assess each gene within the region as a candidate for the PRS component of the phenotype. Clinical array-based comparative genome hybridisation (aCGH) data were used to define a 2.08 Mb minimum region of overlap among four de novo deletions and one mother-son inherited deletion associated with at least one component of PRS. Commonly associated anomalies were talipes equinovarus (TEV), finger contractures and crumpled ear helices. Expression analysis of the orthologous genes within the PRS critical region in embryonic mice showed that the strongest candidate genes were FBN2 and PHAX. Targeted aCGH of the critical region and sequencing of these genes in a cohort of 25 PRS patients revealed no plausible disease-causing mutations. In conclusion, deletion of ∼2 Mb on 5q23 region causes a clinically recognisable subtype of PRS. Haploinsufficiency for FBN2 accounts for the digital and auricular features. A possible critical region for TEV is distinct and telomeric to the PRS region. The molecular basis of PRS in these cases remains undetermined but haploinsufficiency for PHAX is a plausible mechanism.

Exploring microcephaly and human brain evolution

Microcephaly, a commonly documented finding in paediatric neurological practice, confers little diagnostic specificity. Defined by a significant decrease in head circumference, it does not do much to discriminate between the many processes that result in reduced cranial capacity. Given that reduced skull volume reflects the size of the underlying brain, microcephaly is either the consequence of impaired cerebral growth during development or destructive neurological insults. It may be the consequence of environmental, maternal, or genetic aetiologies that include birth asphyxia, intrauterine infections, teratogen exposure, metabolic disease, chromosomal abnormalities, and single gene disorders. Frequently, associated developmental anomalies or neurological findings assist to make specific diagnoses. However, microcephaly may occur in isolation, and when this is extreme (4 or more standard deviations below the population mean), a diagnosis of primary microcephaly (MCPH) is likely. Surprisingly, despite marked reduction in cerebral cortical volume, such patients have mild ⁄ moderate intellectual disability without additional neurological deficits. As an autosomal recessive genetic disorder, this condition has been extensively studied in recent years yielding significant insight into the cellular and regulatory mechanisms governing brain growth. Brain volume is reduced to as little as a third of normal in primary microcephaly. This is accompanied by simplified cortical gyration with an otherwise structurally normal brain on neuroimaging. Over the last 3 million years our brains have seen a three-fold increase in size and 100-fold increase in surface area through cortical folding (gyrification). Evolutionary parallels have consequently been drawn with the expectation that genes identified may provide insight into human brain evolution. Over the last decade seven genes have been identified: MCPH1, WDR62, CDK5RAP2, CEP152, ASPM, CENPJ (CPAP), and STIL. All are expressed in the neuroepithelium during embryonic neurogenesis and localize to the centrosomes and mitotic spindle poles. It is therefore thought that primary microcephaly is a disorder of neurogenic mitosis, where impaired proliferation of neural progenitor cells results in reduced numbers of neurons and a smaller brain. By determining the orientation of the mitotic spindle, these centrosomal proteins may influence the mode of stem cell division, augmenting the stem cell pool and consequently, brain size. This could provide an explanation for how neurogenesis has been modified during primate evolution to achieve expansion of the cerebral cortex. Intriguingly, comparative genomic sequencing has detected adaptive sequence changes in some of the MCPH genes. This raises the possibility that changes in these genes have directly contributed to increasing brain size, though this conjecture remains to be proven. In the last 2 years, the phenotypes associated with the MCPH genes have been substantially extended. Firstly, exome sequencing has identified mutations in WDR62 in a range of severe brain malformations. These include phenotypes usually thought of as distinct diagnostic entities, such as pachygyria, lissencephaly, schizencephaly, polymicrogyria, and cerebellar hypoplasia. Furthermore, the association of mutations in WDR62 with asymmetric polymicrogyria, unilateral cerebrellar hypoplasia, or open-lipped schizencephaly, suggests that such lesions should not be automatically attributed to vascular-disruptive aetiologies. Secondly, MCPH genes have also been associated with microcephalic primordial dwarfism, a group of autosomal recessive disorders with global growth failure. Here, prenatal growth restriction is followed by a marked reduction in postnatal height as well as brain size. Mutations in both CEP152 and CENPJ have been reported but why mutations in the same genes should cause localized reduction in brain size or global growth failure remains unclear. Maintaining the centrosomal theme, pericentrin is also an established and major disease gene for microcephalic dwarfism. However, centrosomes are not the whole story; the recent identification of multiple genes encoding components of the DNA replication machinery and an RNA splicing gene have demonstrated that at least for microcephalic primordial dwarfism other mechanisms are in play. Lastly, progress been made on the most extreme microcephaly phenotype in which profound intellectual disability is accompanied by brain size a tenth of its normal weight. Mutations in the NDE1 gene have been reported by two groups. This gene encodes a centrosomal protein involved in neuronal migration, as well as mitotic spindle formation, these functions respectively explaining the observed partial deficiency in cortical lamination and extreme microcephaly with grossly simplified cortical gyral structure (microlissencephaly). As well as affording insight into neurobiology and evolution, the burgeoning list of disease genes will have diagnostic utility. Comprehensive molecular testing should soon be a realistic possibility, informing diagnosis and patient management as well as permitting families to make informed reproductive choices. Though screening these large genes with established sequencing technologies has been cumbersome, the advent of next-generation sequencing technologies in diagnostic laboratories will provide the realistic prospect of efficient and cost-effective testing to assist the clinician in dissecting the heterogeneous entity that is microcephaly. JENNIE E MURRAY | ANDREW P JACKSON MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK