Diagnostic approach to microcephaly in childhood: a two-center study and review of the literature


  • Maja von der Hagen,

    1. Abteilung Neuropaediatrie, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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  • Mark Pivarcsi,

    1. Department of Pediatric Neurology, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Institute of Neuroanatomy and Cell Biology, Charité – Universitätsmedizin Berlin, Berlin, Germany
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  • Juliane Liebe,

    1. Abteilung Neuropaediatrie, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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  • Horst von Bernuth,

    1. Department of Pediatric Immunology, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Labor Immunologie Berlin, Charité Vivantes GmbH, Berlin, Germany
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  • Nataliya Didonato,

    1. Institut für Klinische Genetik, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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  • Julia B Hennermann,

    1. Department of Pediatric Endocrinology, Gastroenterology and Metabolic Disease, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Villa Metabolica, Department of Pediatrics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
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  • Christoph Bührer,

    1. Department of Neonatology, Charité – Universitätsmedizin Berlin, Berlin, Germany
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  • Dagmar Wieczorek,

    1. Institute of Human Genetics, University Duisburg-Essen, Essen, Germany
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  • Angela M Kaindl

    Corresponding author
    1. Department of Pediatric Neurology, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Institute of Neuroanatomy and Cell Biology, Charité – Universitätsmedizin Berlin, Berlin, Germany
    • Correspondence to Angela M Kaindl, Department of Pediatric Neurology, Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: angela.kaindl@charite.de

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The aim of this study was to assess the diagnostic approach to microcephaly in childhood and to identify the prevalence of the various underlying causes/disease entities.


We conducted a retrospective study on a cohort of 680 children with microcephaly (399 males, 281 females; mean age at presentation 7–8mo, range 1mo–5y) from patients presenting to Charité – University Medicine Berlin (n=474) and University Hospital Dresden (n=206). Patient discharge letters were searched electronically to identify cases of microcephaly, and then the medical records of these patients were used to analyze parameters for distribution.


The putative aetiology for microcephaly was ascertained in 59% of all patients, leaving 41% without a definite diagnosis. In the cohort of pathogenetically defined microcephaly, genetic causes were identified in about half of the patients, perinatal brain damage accounted for 45%, and postnatal brain damage for 3% of the cases. Microcephaly was associated with intellectual impairment in 65% of participants, epilepsy was diagnosed in 43%, and ophthalmological disorders were found in 30%. Brain magnetic resonance imaging revealed abnormalities in 76% of participants.


Microcephaly remains a poorly defined condition, and a uniform diagnostic approach is urgently needed. A definite aetiological diagnosis is important in order to predict the prognosis and offer genetic counselling. Identifying gene mutations as causes of microcephaly increases our knowledge of brain development and the clinical spectrum of microcephaly. We therefore propose a standardized initial diagnostic approach to microcephaly.


Cranial magnetic resonance imaging


Comparative genomic hybridization


Occipitofrontal head circumference

Microcephaly is defined as an occipitofrontal head circumference (OFC) below the third centile or more than 2 standard deviations (SD) below the mean for sex, age, and ethnicity.[1, 2] The term ‘severe’ microcephaly is applied to an OFC more than 3SD below the mean. Microcephaly is associated with a reduction in brain volume and often intellectual and/or motor disabilities. The pathogenesis of microcephaly is heterogeneous, ranging from genetic causes to environmental factors that can have an impact on developmental processes that influence brain size.[3-5] Any condition that affects important processes of brain growth, such as progenitor cell proliferation, cell differentiation, and cell death, can thus induce microcephaly. Anomalies leading to microcephaly may exclusively affect cerebral development (non-syndromal microcephaly) or may be associated with extracranial malformations and/or facial dysmorphism (syndromal microcephaly).

Microcephaly may be evident at birth (primary microcephaly) or postnatally (secondary microcephaly). The child with secondary microcephaly has a normal OFC at birth and then subsequently the relative OFC drops to a value more than 2SD below the mean. These terms do not imply distinct aetiologies. Both primary and secondary microcephaly can be acquired or genetic. The distinction of primary and secondary microcephaly enables clinicians to rank the likelihood of a putative diagnosis according to disease prevalence.

The phenotype of microcephaly is variable and the spectrum of associated disorders is large, with more than 900 entries in the Online Mendelian Inheritance in Man compendium for the clinical sign ‘microcephaly’ as of January 2014. The aim of the present study was to analyse in a large cohort of patients with microcephaly (1) the frequency of (putative) causes of microcephaly; (2) the frequency of structural brain abnormalities, intellectual disability, and associated disorders; (3) the diagnostic steps taken to define the underlying disease; and (4) the number of cases in which the diagnostic approach was successful. We also propose uniform data documentation and a standardized initial diagnostic approach to children with microcephaly.


Patients with microcephaly were recruited from all children who presented to the Departments of Paediatric Neurology at the Charité – University Medicine Berlin and the Dresden University of Technology between 2000 and 2010 and between 2006 and 2011, respectively. Patients were identified by carrying out a computer-based search through patient discharge letters using the terms ‘microcephaly’, ‘developmental delay’, and ‘intellectual disability’, and their corresponding ICD-10 Classification of Mental Disorders numbers.[6] Thus, 680 patients (399 [59%] males, 281 [41%] females) with microcephaly were eligible for our study (Charité – University Medicine Berlin, n=474; Dresden University of Technology, n=206). Eighty-eight families (12%) reported consanguinity.

We reviewed the medical records of all the patients included in our study: medical history, clinical, laboratory, genetic, and radiological data were collected using an anonymous form in a database. All parameters were analyzed for distribution within the entire patient group. Owing to the retrospective nature of our study, not all data were available across the entire cohort. Microcephaly was defined as an OFC below the third centile for sex and age. It was further categorized as primary if it was first apparent at birth or secondary if it occurred postnatally. Proportionate microcephaly was defined as OFC, length, and weight below the third centile for sex and age, whereas disproportionate microcephaly was defined as isolated microcephaly, implying length and/or weight to be above the third centile for sex and age. Cognitive development was assessed using the Bayley Scales of Infant Development, the Wechsler Preschool and Primary Scale of Intelligence, the Wechsler Scale of Intelligence for Children or for Adults, a Snijders–Oomen Non-verbal Intelligence Test, and the Kaufman Assessment Battery for Children or for Adolescents, depending on the age of the child. If results of a specific test were not available, cognitive development was estimated based on the clinical status and the history of the patient (e.g. schooling for children with intellectual disability). Methods of testing regarding the severity of cognitive or motor deficit varied within one institution.


Our cohort comprised 680 patients with microcephaly. There was a predominance of male patients (59% [n=399] male, compared with 41% [n=281] female). The children first presented at a mean age of 7 to 8 months (range 1mo to 5y). Microcephaly was proportional in 42% (n=288) and disproportional in 40% (n=269) of the patients, and classification was not possible in 18% (n=123) of cases. Primary and secondary microcephaly could be differentiated in 42% of patients (n=287), with primary microcephaly occurring in 38% (n=109) and secondary microcephaly in 62% (n=178) of all patients in whom OFC at birth was documented. Among patients with primary microcephaly, there was a slight predominance of individuals with proportional microcephaly (proportional, n=86; disproportional, n=48). We were able to obtain data regarding the gestational age of 433 patients: 64% (n=277) were born at term and 36% (n=156) were born preterm. Severe perinatal complications were reported in 27% (n=183) of the cohort.

Intellectual disability or neurodevelopmental delay was diagnosed in 65% of patients (n=442). We were able to obtain data with regards to schooling for 24% (n=164) of the total cohort. Of these, 28% of the children (n=46) were integrated in mainstream schools and kindergarten and 72% of the children (n=118) received special education. Epilepsy was diagnosed in 43% (n=291) of the patients. Regarding the association between cognitive impairment and the microcephaly classification, the following distribution was noted: (1) 74% (n=214) of 288 patients with proportional and 61% (n=165) of 269 patients with disproportional microcephaly displayed intellectual disability; and (2) cognitive impairment was detected in 34% (n=98) of patients with primary and 57% (n=163) of patients with secondary microcephaly (primary and secondary microcephaly could be specified in 287 patients). Our data analysis revealed that in 18% of the cases proportional microcephaly was not differentiated from disproportional microcephaly, and in 58% of the cases microcephaly was not classified into primary or secondary microcephaly during the diagnostic work-up. This made it impossible to retrospectively define the microcephaly phenotype of a patient.

Cranial magnetic resonance imaging (cMRI) or cerebral ultrasound was performed in 72% (n=491) of our cohort at a mean age of 23 months. The results were abnormal in 63% (n=310) of cases. Of the 299 children who were assessed by cMRI, abnormal findings were found in 76% (n=227). The most frequent structural brain lesions, apart from microencephaly, were anomalies of the white matter, found in 40% (n=90), and gyration defects, found in in 14% (n=31) of all radiologically assessed patients. White matter abnormalities included periventricular leukomalacia and delayed or disturbed myelination. Further frequent structural brain lesions included corpus callosum anomalies in 31% (n=70) and anomalies of the cerebellum in 15% (n=33) of the radiologically examined children.

Non-central nervous system abnormalities and malformations associated with microcephaly were revealed through medical history, physical examinations, and further tests. Disorders of the eyes were diagnosed in 30% (n=207) of the patients, of the ears in 8% (n=51), of the heart in 14% (n=93), of the kidneys and the urinary tract in 13% (n=89), and of the gastrointestinal tract in 9% (n=58). Other disorders included facial dysmorphism in 19% (n=127) of patients, and anomalies of the oropharynx such as cleft palate in 13% (n=87), of the skeletal system in 13% (n=91), of the skin in 2% (n=12), and of the hair in 1% (n=9) of the patients.

The putative aetiology of microcephaly was documented in 59% of the patients (n=403). Among the total cohort of 680 patients, pathogenesis was genetic or presumably genetic in 29% (n=194). Other causes of microcephaly included craniosynostosis in 2% (n=14), perinatal brain injury in 27% (n=182), and postnatal brain injury in 2% (n=13). In 41% the aetiology remained unclear. Genetic abnormalities were numerical chromosome aberrations or microdeletions/duplications in 24% (n=46) and monogenic disorders in 30% (n=58); the remaining diagnoses were putative genetic syndromes based on the patient phenotype and/or the family constellation (consanguinity and/or several family members exhibiting a similar phenotype; Table 1).

Table 1. Causes of microcephaly in our cohort (n=680)
Microcephaly cause n %
  1. a

    Phenotype such as mitochondriopathies, family constellation such as consanguinity, and several affected children.

1. Genetic cause 194 28.5
Microcephaly syndromes with numerical chromosomal aberrations or microdeletion and/or duplication syndromes 46 6.8
Microdeletion and/or duplication syndromes28 
Trisomy 11/221 
Patau syndrome, trisomy 131 
Down syndrome, trisomy 214 
Pallister–Killian syndrome, tetrasomy 12p1 
Unbalanced deletion of chromosome 12/duplication of chromosome 171 
Pitt–Hopkins syndrome, deletion 18q21.21 
Microdeletion 22q112 
Klinefelter syndrome3 
Triple X syndrome2 
Monogenetic microcephaly syndromes/diseases 58 8
with autosomal dominant inheritance 13  
Cornelia de Lange syndrome2 
Werner syndrome1 
Currarino syndrome1 
Charcot–Marie–Tooth disease 1A1 
Kabuki syndrome2 
Mowat–Wilson syndrome1 
Generalized epilepsy with febrile seizures (SCN1A)1 
Glucose transporter type 1 deficiency (GLUT1)1 
Tuberous sclerosis1 
Congenital Rett syndrome (FOXG1)2 
with autosomal recessive inheritance 17  
Primary autosomal recessive microcephaly 12 
Nijmegen breakage syndrome1 
Nijmegen breakage syndrome-like disorder (RAD50)1 
Marinesco–Sjögren syndrome1 
Warburg micro syndrome1 
Congenital muscular dystrophy with α-dystroglycan deficiency (POMT1)2 
Metachromatic leukodystrophy1 
Niemann–Pick disease type C1 
Dyggve–Melchior–Clausen disease1 
Cohen syndrome1 
Batten disease1 
Carnitine palmitoyltransferase IA deficiency (CPTIA)1 
Methylenetetrahydrofolate reductase deficiency (MTHFR)1 
3-Methylcrotonyl-CoA Carboxylase 1 deficiency (MCCC1)1 
with X-chromosomal inheritance 21  
Rett syndrome (MECP2)13 
Duchenne muscular dystrophy2 
Becker muscular dystrophy1 
Léri–Weill syndrome1 
Pelizaeus–Merzbacher disease1 
Menkes syndrome1 
Allan–Herndon–Dudley syndrome (MCT8)1 
Incontinentia pigmenti1 
With complex inheritance/not classified7 
Angelman syndrome6 
Prader–Willi syndrome1 
2. Putative genetic cause due to phenotype or family constellation a 90 13
3. Perinatal brain injury 182 26.7
Birth complications118 
Maternal disease during pregnancy25 
Exposure to teratogen substances30 
Other pregnancy disorders9 
4. Postnatal brain injury 13 1.9
Non-accidental injury (‘battered child’)3 
5. Craniosynostosis 14 2.1
6. Unknown cause 277 40.7


Evaluation of a child with microcephaly

In the current retrospective study, we assessed the diagnostic approach to patients with microcephaly and identified the prevalence of various underlying causes of the disorder. Our cohort is, to our knowledge, the largest cohort studied so far, with most previously published cohorts staying well below 100 cases.[7-14] Males predominated among patients with microcephaly presenting to our clinical centres (59% males [n=39] compared with 41% females [n=281]); the distribution of proportional and disproportional microcephaly was almost equal, but there was a predominance of secondary (postnatal) microcephaly relative to primary (congenital) microcephaly (62% vs 38% of the 287 patients in whom type of microcephaly was specified). The difference between affected males and females might be explained by mutations in X-chromosomal recessive genes, which primarily affect males.

Phenotype evaluation and microcephaly classification

The majority of children with microcephaly presented with neurological symptoms in infancy at a mean age of 7 to 8 months. Neurodevelopmental delay or intellectual disability was the most frequent reason for referral to our departments (65% of all cases), and epilepsy was another common reason (43% of all cases). This is in line with the data of Abdel-Salam et al.,[15] who reported an overall prevalence of epilepsy in children with microcephaly of 40.9%. Epilepsy seems to be more common in children with secondary microcephaly than in those with primary microcephaly.[15, 16] In addition, microcephaly has been identified as a risk factor for intellectual disability and therapy-refractory epilepsy.[13, 17, 18] Among children with neurodevelopmental delay, secondary microcephaly is more common than primary microcephaly (57% vs 34% of patients with classified microcephaly; n=287). In this retrospective data analysis of children with intellectual disability, proportional microcephaly was more common than disproportional microcephaly (74% vs 61% of patients with classified microcephaly; n=557). Whether proportional microcephaly is more predictive of developmental delay than disproportional microcephaly is an open question as the available data are conflicting.[18]

In our cohort, of the 164 children for whom data could be obtained with regards to schooling, 72% did not attend mainstream schools or kindergarten but rather needed special education. It needs to be noted here that children examined at the paediatric neurology departments represent a selective cohort of mainly symptomatic patients with microcephaly, and thus it is difficult to estimate the prevalence of children with microcephaly and normal psychomotor development.

Further clinical findings frequently identified in patients with microcephaly were ophthalmological disorders (30%), facial dysmorphism (19%), anomalies of the oropharynx including cleft palate (13%), and anomalies of the heart (14%), kidneys and of the urinary tract, as well as of the skeletal system (13% each) and of the gastrointestinal tract (9%). This emphasizes the need for a multidisciplinary approach to patients with microcephaly. Ophthalmological and audiological disorders and complex ear anomalies have been associated in variable prevalence with microcephaly,[13] and, therefore, the patients with microcephaly of unknown aetiology require screening and follow-up monitoring.

In nearly a third of the patients, the underlying disease could be diagnosed without further extensive diagnostic work-up or with focused genetic testing based on patient history revealing intrauterine brain damage (e.g. drug abuse), or severe perinatal complications (e.g. severe asphyxia), or on presentation with a typical phenotype of a known syndrome (e.g. Down syndrome). We detected a large degree of variability in the data available for individual patients and therefore conclude that a standardized assessment of medical history, clinical examination, and performed studies is urgently required. We suggest using the term ‘primary microcephaly’ instead of ‘congenital microcephaly’ and ‘secondary microcephaly’ rather than ‘postnatal microcephaly’ and clearly defining whether a microcephaly is proportional or disproportional. The exact classification can direct future diagnostic investigations and potentially allow a prognosis. Even in a university-based paediatric neurology setting the diagnostic approach and documentation is not standardized or uniform, and in our cohort microcephaly was of unknown origin or unclassified in a high percentage of cases.


cMRI or cerebral ultrasound was performed in 72% of all children with microcephaly, and the majority of these patients underwent imaging by the second year of life (the mean age at which cMRI was performed was 23mo, and cranial ultrasound was performed in the first months of life). Children in whom cMRI was performed before 24 months of age often underwent repeated MRI studies to assess myelination after 24 months, by which age most myelination is complete. The results were abnormal in 63% of the patients. Of the 299 children who were assessed by cMRI, abnormal findings in addition to microcephaly were found in 76%. The higher prevalence of abnormal findings in cMRI analyses points to cMRI as the more sensitive imaging method for the identification of brain lesions and anomalies associated with microcephaly. Abnormal findings in addition to microcephaly comprise white matter anomalies (40%), corpus callosum anomalies (31%), infratentorial lesions (15%), and gyration defects (14%). Recent neonatal and prenatal imaging studies suggest that agenesis of the corpus callosum occurs at least 1:4000 live births, and other imaging studies have demonstrated that 3% to 5% of individuals assessed for neurodevelopmental disorders have agenesis or hypoplasia of the corpus callosum.[19-21] Agenesis of the corpus callosum can have genetic causes or result from various exogenous factors such as infectious, vascular, or toxic effects. White matter anomalies were, as expected, particularly frequent in preterm infants. MRI has been reported to be valuable in the evaluation of children with developmental delay and at least one further neurological sign, including atypical head circumference.[22] Jaworski et al.[23] found that the percentage of imaging abnormalities was highest in patients with microcephaly and a known history of perinatal or postnatal brain injury (91%). The proportion of patients with microcephaly and one or more extracranial congenital anomalies in whom imaging abnormalities were detected was somewhat lower (67%).[23]

Although most of the radiological findings were rather unspecific and did not enable a specific diagnosis, in some cases they directed the diagnostics towards further metabolic screening or genetic testing. In some patients, the identification of a specific pattern of brain injury enabled the attending physician(s) to refrain from further diagnostic work-up. The small percentage of children in whom the cMRI finding led to specific genetic testing and enabled a diagnosis included those with autosomal recessive primary microcephaly, lissencephaly and further gyration defects, Pelizaeus–Merzbacher disease, mitochondriopathies, and tuberous sclerosis.

Genetic assessment

Genetic diagnostics were performed in 51% (n=308) of all patients; diagnostic tests included karyotyping, array comparative genome hybridization (array-CGH) analysis, chromosomal breakage analysis, and sequencing of selected genes. Of all patients in whom the cause of the microcephaly was suspected to be genetic, the genetic abnormality was determined through karyotyping in 2% to 3% of cases and, in recent years, also through array-CGH analysis in about 4% of all patients (this rate is likely to rise further in the years to come in Germany as a result of the increasing application of array-CGH analyses in the routine diagnostic work-up and of exome sequencing in the research setting for intellectual disability). In many patients the initial tests produced normal results and thus were subsequently often followed by more specific/directed genetic analysis. Through this approach, a specific genetic cause was identified in 15.3% of all patients (n=104): numerical chromosome aberrations and microdeletions/duplications in 6.8% (n=46) and monogenic disorders (e.g. Rett syndrome, Angelman syndrome) in 8.5% (n=58; Table 1). Our approach thereby follows current recommendations for the diagnostic work-up for intellectual disability.[24] According to the proposed diagnostic algorithm, in the investigations of neurodevelopmental delay, array-CGH analyses present the next diagnostic step.[24]

In a subgroup of patients with microcephaly, a genetic aetiology is strongly suspected owing to phenotype and/or family history (further family members affected, consanguineous parents), even if karyotyping, array-CGH, and sequencing of selected genes are not conclusive. In this group, whole-exome or whole-genome sequencing may allow for the identification of the underlying genetic abnormality. The possibilities for genetic analyses have changed dramatically within the last decade, and microarray analyses have become a criterion standard. Next-generation sequencing methods are likely to clarify the underlying cause in patients in whom the aetiology of microcephaly is unknown. However, dysmorphological evaluation is important for the diagnosis of children with syndromes.

Aetiology of microcephaly and the success rate of the approach to its diagnosis

The aetiology of microcephaly is highly variable and heterogeneous. In our study, genetic causes accounted for 29% (n=194) of all patients with microcephaly, followed by perinatal brain injury in 27% (n=182), postnatal brain injury in 2% (n=13), and craniosynostosis in 2% (n=14). In 41% of patients the aetiology remained unclear. Inborn errors of metabolism, e.g. mitochondriopathy, Menkes disease, and methylenetetrahydrofolate reductase deficiency, were counted as genetic causes. They occurred in about 3% of the total cohort and rarely resulted in non-syndromal (isolated) congenital microcephaly. The exact prevalence of inborn errors of metabolism among children with microcephaly is unknown. However, based on previous studies of children with global developmental delay, it is likely to be from 1% to 5%, similar to our finding.[13, 25-28]

The underlying cause of microcephaly in our cohort was identified in 59% of cases (n=403), meaning that the families of 41% (n=277) of patients may carry the burden of not knowing the reason for their child's microcephaly. In these cases, the utility of genetic and clinical counselling is limited. In our cohort, perinatal brain injury accounted for a large proportion (27%) of the patients with microcephaly (n=181), many of whom were born preterm. Both departments of paediatric neurology are associated with large neonatal intensive care units that offer the highest level of care and are involved in the neurodevelopmental follow-up of children. This may also account for the high rate of perinatal brain damage in our cohort. Furthermore, it needs to be noted here that an underlying genetic cause may have caused preterm birth and rendered a patient more susceptible to, or mimicked, perinatal damage. A comprehensive history, growth records for the child and the close family, and a detailed physical examination may suggest a diagnosis or direct further testing.

In summary, our data show that microcephaly is still poorly defined and that the diagnostic approach in children with microcephaly is not uniform. In a large subgroup, it was not possible to make a definite diagnosis using the current approach. We, therefore, propose uniform data documentation and a standardized initial diagnostic approach to a child with microcephaly.

Diagnostic algorithm for the initial evaluation of paediatric microcephaly

A standardized, evidence-based, algorithmic approach is needed for the rapid identification of frequent causes of microcephaly as well as rare diseases, which can later be studied in research projects in order to decipher the phenotype and pathomechanism of genetically defined diseases. We have developed a standard questionnaire that can be used to document the patient's own history and family history, the clinical status (Fig. S1, online supporting information), and further diagnostic work-up results in the case that history and clinical evaluation do not identify the underlying cause (Fig. S2, online supporting information). Based on our data and previously published data, we further propose a common initial approach to children with microcephaly (Fig. 1).

Figure 1.

Diagnostic approach to a child with microcephaly. Based on our data and previously published data, we further propose a common initial approach to a child with microcephaly. CGH-array, comparative genomic hybridization array; MRI, magnetic resonance imaging; CMV, cytomegalovirus.

Standardized assessment of medical history and physical examination

The first diagnostic step towards identifying a child with microcephaly is gathering a comprehensive medical and family history and collecting detailed clinical examination data. For the purpose of standardization, we propose a questionnaire in which the relevant data can be recorded, including the age at onset, severity, family history (pedigree), and putative causes such as perinatal brain damage, metabolic diseases, and genetic causes (Fig. S1).

In the questionnaire we have adopted the microcephaly terminology as stated in the introduction: primary microcephaly evident at birth and secondary postnatal, proportionate and disproportionate microcephaly depending on OFC, weight, and height of a patient. We are, of course, aware of the numerous additional microcephaly terminologies used in the literature, but these are, in our view, unnecessary and hamper a standardized diagnostic approach. The distinction of primary and secondary microcephaly enables clinicians to rank the likelihood of a putative diagnosis according to timing of microcephaly occurrence in individual disease entities, although for many diseases the exact classification of microcephaly has not been reported. The classification of microcephaly into proportionate or disproportionate microcephaly is important, as identification of proportionate microcephaly should, e.g., prompt diagnostic steps with respect to dystrophy in an infant or toddler.

The OFC should be measured several times with a non-elastic measuring tape, and values should be plotted in a centile or SD curve parallel to the other anthropometric data. In utero head circumference can provide a rough estimate of the time of the first deviation from the norm in primary microcephaly. Frequent OFC measurements early in life are justified by the high rate of brain growth in the first 3 years.[29] Measuring parental OFCs is essential for the diagnosis of familial microcephaly. OFCs are, unfortunately, often plotted in curves based on older values taken from studies by Nellhaus[30] or Prader et al.[31] For children in industrial countries, the mean OFC is larger than that indicated in the WHO standard values, which are based on measurements taken from about 8500 children in Brazil, Ghana, India, Oman, and the USA (www.who.int/childgrowth/en). We thus recommend using the growth charts published by the Centers for Disease Control and Prevention (CDC; www.cdc.gov/growthcharts/). In several Anglo-Saxon countries, the OFC is evaluated in SDs, and this can be calculated based on the values for the mean and SD given on the CDC site.

In a child with primary or early infantile secondary microcephaly, exogene factors that can damage the developing brain during pregnancy and perinatally should be assessed in detail. Such factors include prenatal infections, prenatal exposure to hypoxia, radiation, toxins, medications or drugs of abuse, preterm birth, and maternal disease.

The last part of the questionnaire provides a comprehensive overview of important aspects of the clinical assessment of a child with microcephaly, including a neuropsychological evaluation. The latter is of particular interest as children with microcephaly carry a higher risk of developmental delay than their peers with normocephaly.[13, 14, 18] The aim of the detailed clinical examination is to pinpoint the neurological phenotype and identify signs of major or minor abnormalities potentially leading to the diagnosis of a syndromal microcephaly. This basic information can be collected without high costs or further testing and may help to resolve many of the potential aetiologies of microcephaly in a child. Moreover, further diagnostic work-up may not be indicated in a child with microcephaly, but normal neurocognitive and motor development, and no further signs of an underlying disease.

The combination of knowledge on disease prevalences and the accumulated information obtained from the history and detailed physical examination may already suggest a specific diagnosis and direct diagnostic testing. This was the case in one-third of the patients in our cohort. For example, in a female patient, typical development in the first months of life followed by development of secondary microcephaly, loss of acquired skills, occurrence of intellectual disability, and stereotypic movements will prompt molecular genetic analysis of the MECP2 gene associated with Rett syndrome. Similarly, a history of maternal alcohol abuse during pregnancy, typical facial dysmorphism, and extracranial symptoms may lead to the diagnosis of a fetal alcohol spectrum disorder, and a history of immunodeficiency, cancer, and microcephaly may hint towards the work-up of chromosomal breakage syndromes. An overview of causes for primary and secondary microcephaly is given in Tables 2 and 3.

Table 2. Causes of primary microcephaly: overview
1. Genetic causes
Numerical chromosomal aberrations or microdeletion and/or duplication syndromes
Trisomy 13, 18, 21 etc.
Monogenetic microcephaly
Autosomal recessive microcephaly (MCPH1-10, MCPHA)
Nijmegen breakage syndrome (MIM#251260)
Autosomal dominant microcephaly
X-chromosomal microcephaly
Aicardi–Goutières syndrome (MIM#225750, 610329, 610181, 610333, 612952)
Cockayne syndrome (MIM#216400, 133540, 216411)
Cornelia de Lange syndrome (MIM#122470, 610759, 614701, 300590, 300822)
Rubinstein–Taybi syndrome (MIM#180849)
Feingold syndrome (MIM#164280, 614326)
Rett syndrome, congenital (MIM#164874)
Mowat–Wilson syndrome (MIM#235730)
Smith–Lemli–Opitz syndrome (MIM#270400)
Seckel syndrome (MIM#210600, 606744, 608664, 613676, 613823, 61472)
Ligase IV syndrome (MIM #606593)
Mutations in ATRX gene (MIM*300032)
Mutations in ARX gene (MIM*300382)
Mutations in PQBP1 gene (MIM*300463)
Mutations in ASNS gene (MIM*108370)
Borjeson–Forssman–Lehmann syndrome (MIM#301900)
Imprinting disorders
Angelman syndrome (MIM#105830)
2. Metabolic cause (genetic aetiology)
Serine biosynthesis disorder
Sterol biosynthesis disorder
Mitochondriopathy, e.g. pyruvate dehydrogenase deficiency
Congenital disorders of glycosylation syndrome
Rare congenital metabolic diseases (see text)
3. Exogenic factors
Intrauterine infection
Toxoplasmosis, rubella, cytomegalovirus, herpes simplex, varicella zoster virus, syphillis, human immunodeficiency virus
Alcohol, cocaine, antiepileptic drugs, lead/mercury intoxication, radiation
Disruptive incident
Vascular incident (stroke), intrauterine death of twin
Maternal disease
Maternal anorexia nervosa
Extreme insufficiency of placenta
4. Craniosynostosis
Table 3. Causes of secondary microcephaly: overview
1. Genetic causes
Numerical chromosomal aberrations or microdeletion and/or duplication syndromes
e.g. Williams syndrome
Monogenetic microcephaly
Aicardi–Goutières syndrome (MIM#225750, 610329, 610181, 610333, 612952)
Ataxia telangiectasia (MIM*607585)
Cohen syndrome (MIM#216400)
Early infantile epileptic encephalopathies, e.g. EIEE14 (MIM#614959)
Marden syndrome (%248700)
Mowat–Wilson syndrome (MIM#235730)
Rett syndrome (MIM#312750)
Rubinstein–Taybi syndrome (MIM180849)
Imprinting disorders
Angelman syndrome (MIM#105830)
2. Metabolic causes (genetic aetiology)
Phenylketonuria (untreated)
Glycine encephalopathy
Disorders of serine biosynthesis
Urea cycle disorders
Disorders of cobalamin metabolism
Organic aciduria
Disorders of neurotransmitters and biogenic amines
Congenital disorders of glycosylation syndrome (CDG)
Glucose transporter defect (GLUT1)
Leukodystrophies, e.g. Pelizaeus–Merzbacher diseases
Menkes diseases
Neuronal ceroid-lipofuscinosis (NCL)
Peroxisomal disorders
Disorders of sterol biosynthesis
Disorders of purine and pyrimidine metabolism
Molybdenum cofactor deficiency and sulphite oxidase deficiency
Disorders of pyridoxine metabolism
Lysosomal storage disorders, e.g. mucolipidosis, multiple sulphate deficiency, Gaucher disease type 11
3. Exogenic factors
Perinatal brain damage
Hypoxic–ischaemic encephalopathy
Perinatal infection, e.g. herpes simplex virus, rubella virus, and syphilis (if acquired in third trimester)
Perinatal/postnatal haemorrhagic and ischaemic insult
Perinatal intracranial haemorrhage/thrombosis
Perinatal teratogens
Postnatal brain damage
Infections, e.g. meningitis, encephalitis
Intracranial haemorrhage/thrombosis
Traumatic brain injury, intracranial haemorrhage
Haemorrhagic and ischaemic insult
Psychosocial deprivation
Toxic, e.g. lead toxicity, uraemic encephalopathy (renal insufficiency)
Endocrinology, e.g. hypothyroidism, hypopituitarism
Chronic or systemic disorders, e.g. congenital heart disease, anaemia
Vitamin B12 deficiency in fully breastfed infant of vegan mothers
Structural brain anomalies
E.g. holoprosencephaly. May also be of genetic aetiology, e.g. HPE1–9
4. Craniosynostosis

Approach to a child with microcephaly

In those individuals in whom the diagnosis remains unclear following a comprehensive medical history and clinical examination, we suggest MRI of the brain as the next step. Although the majority of radiological findings were unspecific in our cohort and did not enable a specific diagnosis, they did direct further diagnostic measures. For example, white matter disease indicative of leukodystrophy on cMRI will tend to provoke metabolic investigations, whereas certain brain malformations, such as lissencephaly, lead first to genetic testing.

Inborn errors of metabolism more often lead to secondary microcephaly than to primary microcephaly. In secondary microcephaly, metabolic investigations should be performed, as indicated in Fig. 1. Routine metabolic screening of all patients with microcephaly is not required, but targeted metabolic studies should be performed based on findings in the patient's medical and family history, clinical examination, and neuroimaging. In the case of leukodystrophy in particular, extensive metabolic and enzymatic diagnostic work-up should be initiated. Some inborn errors of metabolism that are associated with an accumulation of toxic metabolites or an intrauterine lack of metabolites may lead to intrauterine brain damage and subsequently to primary microcephaly (Table 4). These diseases include serine biosynthesis disorders (e.g. 3-phosphoglycerate dehydrogenase deficiency, phosphoserine phosphatase deficiency), which are associated with further neurological symptoms such as muscular hypotonia and epilepsy and are diagnosed by analyses of amino acids in blood and cerebrospinal fluid.[32] Smith–Lemli–Opitz syndrome, a disorder of sterol biosynthesis, should be suspected when patients show typical clinical signs such as minor facial anomalies, syndactyly, and organ malformations.[33] Furthermore, mitochondrial disorders (e.g. pyruvate dehydrogenase defect, respiratory chain defect, mitochondrial transporter defect)[34-36] and congenital disorders of glycosylation (i.e. CDG-Id, -Ig, -Ik, -Ilc, -Ile)[37-40] should be tested in patients with primary microcephaly. In addition, there are a few inborn errors of metabolism which are only very infrequently associated with microcephaly at birth (disorders of cobalamin metabolism such as the CbIC or CbIF deficiency)[41, 42] and a few inborn errors of metabolism that are very rare but frequently associated with primary microcephaly (e.g. multiple sulphatase deficiency, congenital neuronal ceroid lipofuscinosis, leukotriene C4 synthesis defect)[43-45] that need to be considered in the differential diagnosis. Patients with molybdenum cofactor deficiency or sulphite oxidase deficiency are mostly normocephalic at birth but develop secondary microcephaly rapidly within the first weeks of life.[46] None of these described inborn errors of metabolism can be identified by the established newborn infant screening programmes.

Table 4. Overview of most common congenital metabolic diseases associated with primary microcephaly
DiseaseAssociated symptomsDiagnostic tests
Maternal phenylketonuriaIntrauterine growth retardation, facial dysmorphism, congenital heart defect, intellectual disabilityPhenylalanine in maternal blood sample
Serine biosynthesis disorderMuscular hypotonia, epilepsy, intellectual disabilityFasting amino acids in plasma and cerebrospinal fluid
Sterol biosynthesis disorderFacial dysmorphism, syndactylia, organ malformation, midline defects, genital malformations, adrenal insufficiency, intellectual disabilitySterol analysis (7/8-dehydrocholesterol) in plasma
MitochondriopathyLactic acidosis, further organ manifestation (e.g. encephalopathy, myopathy, cardiomyopathy, cerebral changes on magnetic resonance imaging)Lactate and pyruvate in blood and cerebrospinal fluid, organic acids in urine, further tests including enzymatic/genetic analyses depend on clinical signs
Congenital disorders of glycosylationBroad clinical picture including intellectual disability, dysmorphism, epilepsy, muscular hypotoniaIsoelectric focusing analysis of transferrin in serum

If the result of cMRI is normal or changes are unspecific, i.e. do not suggest metabolic disease, our algorithm follows current recommendations for the diagnostic work-up of intellectual disability.[24] In this case, array-CGH analysis presents the second diagnostic step.[24] Recent studies point to an average diagnostic yield of chromosomal microarray analyses of 15% to 20% in patients with intellectual disability.[47-49] In the case of autosomal recessive primary microcephaly, i.e. non-syndromal primary microcephaly (OFC <3SD) and intellectual disability without further severe neurological abnormalities, analysis of the most common autosomal recessive primary microcephaly genes such as WDR62 and ASPM using Sanger sequencing or panel diagnostics (next-generation sequencing) is indicated.[50] In syndromal microcephaly, syndromologists should be consulted in making a diagnosis, as they may be able to guide genetic analysis effectively.

In the remaining subgroup of microcephaly patients in whom genetic aetiology is strongly assumed and in whom karyotyping, array-CGH, and Sanger sequencing of the selected genes or next-generation panel sequencing are inconclusive, large screening tests of disease-associated genes and exome or genome sequencing may identify the underlying genetic defect.


Microcephaly is a frequent clinical sign common in many rare diseases. An exact diagnosis is important for counselling of the patient and the affected family regarding clinical course, possible complications, optimized medical support, and recurrence risk. Moreover, an exact diagnosis is also important for future gene therapy approaches and the development of neuroprotective therapies of perinatal brain injuries. If currently available diagnostic tools cannot establish a specific (genetic) diagnosis, modern technologies that are still being optimized at a research level might provide a future diagnostic approach and become part of the standard approach to patients with microcephaly.


The authors thank Christoph Hübner, Theodor Michael, Denise Horn, Birgit Spors, Gabriele Hahn, Rainer John, Sigrid Tinschert, and Pierre Gressens for discussions. Our research was supported by the Charité – Universitätsmedizin Berlin, the Technische Universität Dresden, the German Research Foundation (DFG, SFB665), the BIH (Berlin Institute of Health, Helmholtz Stiftung), the Sonnenfeld Stiftung, and the Deutsche Gesellschaft für Muskelkranke (DGM). The authors state that they had no interests that might be perceived as posing conflict or bias.