METHODS

Fifty-six consanguineous families of northern Pakistani origin containing one or more members with MCPH were ascertained. Families were referred to the authors from two sources: from paediatricians in Yorkshire, UK (serving a large northern Pakistani population centred upon Bradford) and from paediatricians and Special Schools in Rawalpindi, Islamabad, and Lahore in Pakistan (which is the geographical origin of the majority of Asians in Yorkshire). One of the authors obtained a medical history and family pedigree and examined all potentially affected subjects. Ethical Committee consent for this study has been given.

Family members gave blood samples from which DNA was obtained by standard methods. For each MCPH locus, a panel of microsatellite markers was devised (table 1). Each of the 56 families was genotyped against the five known MCPH loci using these panels. As all of the families are consanguineous, identity by descent could be sought and used to assess evidence of linkage to a particular locus.¹¹

RESULTS

In the 56 study families, there were 131 MCPH affected members with a male to female ratio of 70:61. Of these families, 20/56 contained affected subjects in more than one sibship, 22/56 were a single sibship with multiple affected subjects, and in 14/56 a single offspring was affected. All affected subjects were microcephalic at birth with a head circumference between –4 and –14 SD (fig 1). In fifteen of them, the degree of microcephaly at birth could be compared to that at 5-10 years of age and no difference was seen, implying that this is not a progressive condition. There was no sex difference in the degree of microcephaly or the gross cognitive ability. Within multiply affected families, head circumferences usually varied only by ± 1 SD. Where there was a greater variation in head circumference, this was confined to older affected members (>30 years of age) of the family, who had a reduced relative head circumference compared to their younger affected family relatives. All study subjects enjoyed good health, comparable to their unaffected family members. All had normal early motor milestones but some had delay in speech (in acquisition of vocabulary and grammar but without dysarthria), none had severe or profound mental retardation, and none had epilepsy. Most were physically able and the majority were personable, biddable, and trainable in life skills. Some had “sloping foreheads”, but within a family this was an inconsistent feature.¹ All parents were of normal physical and mental ability and none had microcephaly.

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Figure 1

Head circumference measurements of all affected subjects shown as standard deviation from norms by age and sex. Each point represents one or more persons with a diagnosis of autosomal recessive primary microcephaly. The head circumference as a standard deviation from the age and sex norm is shown on the y axis.^12–14 The age of the subjects is shown in years on the x axis. The sex is shown as a circle for a male and a square for a female.

The genotype results are reported in table 2: 2/56 families were consistent with linkage to MCPH1 (4%), 10/56 to MCPH2 (18%), 2/56 to MCPH3 (4%), none to MCPH4, and 24/56 to MCPH5 (43%). In two multi-affected families likely to be linked to MCPH5, a subject was homozygous for a smaller region while carrying the larger family haplotype. We have used these results to refine this locus further. One singleton was homozygous for all markers for both the MCPH2 and MCPH3 loci. In 18/56 families (32%), there was no definite evidence of linkage to one of the five known loci. However, three of these families had affected subjects with a concordant haplotype for MCPH3 markers (that is, the same heterozygous allele results for all polymorphic microsatellite markers assessed) suggesting the possibility of linkage to a smaller homozygous region contained within this haplotype.

Bearing in mind the possibility of a child of first cousin parents carrying a homozygous region by descent rather than as the disease allele is 1 in 16, whereas in multi-affected pedigrees this chance decreases, and that clinical misdiagnosis must always be considered in a “diagnosis of exclusion” such as MCPH, and such phenocopies are more likely in single affected families,³ then, excluding singletons from the analysis, 2/42 families are compatible with linkage to MCPH1 (5%), 6/42 to MCPH2 (14%), 1/42 to MCPH3 (2%), none to MCPH4, 19/42 to MCPH5 (45%), and 14/42 families (33%) did not segregate with any of the loci. This suggests that inclusion of singletons in our analysis was not a major cause of error. All individual head circumferences and the mean for each family were analysed against the MCPH loci genotype data and there was no discernable difference (table 3).

DISCUSSION

Given the large number of disorders that cause “mental retardation with microcephaly”, it is important from a clinical perspective to attempt to split such a broad phenotypic category into more discrete entities. To this end we report a large cohort of subjects in whom we have made a clinical diagnosis of autosomal recessive primary microcephaly (MCPH). Supporting the definition of MCPH as a clinical neurodevelopmental disorder is both the numbers of cases we have amassed and the fact that genetic linkages for MCPH loci have been identified. We suggest that autosomal recessive primary microcephaly may be a more exact and useful term than others in use, for example, “true microcephaly”, “recessive microcephaly”, or “microcephaly vera”.³ Certainly, in our study population the “true microcephaly” phenotype with the sloping forehead was not a consistent finding in subjects affected with MCPH. It has also been suggested that carriers of MCPH have diminished intelligence as a heterozygote effect,¹⁵ though we found no evidence of this.

In conclusion, we suggest that MCPH can be defined as a clinical entity. It was considered that it would exhibit genetic heterogeneity^16,17 and this has now been confirmed, with five MCPH loci reported to date.² We have established the relative incidence of each locus in the northern Pakistani population and that MCPH5 is the most common in this ethnic group. Interestingly, none of our northern Pakistani families link to the MCPH4 locus,¹⁰ which was identified in a Turkish population. Following genotyping of the study families, we were unable to differentiate clinically between families linked to the five MCPH loci. We have also shown that further MCPH loci await discovery with a number of families as yet unlinked. It seems likely that determination of the MCPH genes will give valuable insights into normal brain development (and possibly the evolutionary process of encephalisation, where the surface area of the cerebral cortex has tripled in size from primates to man¹⁸), as well as providing new therapeutic options for affected families.

NOTE ADDED IN PROOF

The MCPH1 gene has now been identified.¹⁹