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Mutations in the Pericentrin (PCNT) Gene Cause Primordial Dwarfism

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Science  08 Feb 2008:
Vol. 319, Issue 5864, pp. 816-819
DOI: 10.1126/science.1151174

Abstract

Fundamental processes influencing human growth can be revealed by studying extreme short stature. Using genetic linkage analysis, we find that biallelic loss-of-function mutations in the centrosomal pericentrin (PCNT) gene on chromosome 21q22.3 cause microcephalic osteodysplastic primordial dwarfism type II (MOPD II) in 25 patients. Adults with this rare inherited condition have an average height of 100 centimeters and a brain size comparable to that of a 3-month-old baby, but are of near-normal intelligence. Absence of PCNT results in disorganized mitotic spindles and missegregation of chromosomes. Mutations in related genes are known to cause primary microcephaly (MCPH1, CDK5RAP2, ASPM, and CENPJ).

The growth of an individual depends on regulation of cell size and cell division. Dysfunction of these regulatory pathways not only results in somatic undergrowth but contributes to a wide variety of pathological conditions, including cancer and diabetes (1). To identify potential regulators of human growth, we used positional cloning to determine the underlying defect in a rare autosomal recessive disorder characterized by extreme pre- and postnatal growth retardation, namely, microcephalic osteodysplastic primordial dwarfism type Majewski II [MOPD II, Mendelian Inheritance in Man (MIM) 210720].

Individuals with MOPD II have an average birth weight of less than 1500 g at term, an adult height of about 100 cm, and a variety of associated bone and dental anomalies (Fig. 1) (2, 3). Despite the small head size (average postpubertal head circumference of 40 cm), brain development appears grossly normal with only a few individuals displaying serious mental retardation, a feature that sets MOPD II apart from primary microcephaly and Seckel syndrome. Far-sightedness, irregular pigmentation, truncal obesity, and type 2 diabetes with onset at or before puberty have been noted in older individuals with MOPD II, and life expectancy is reduced because of a high risk of stroke secondary to cerebral vascular anomalies, often classified as Moyamoya disease (2, 4). Although these features led investigators to hypothesize that MOPD II is a premature aging syndrome (5), we found no evidence of accelerated telomeric shortening as a potential cellular explanation of premature aging in lymphocyte samples of two unrelated female patients with MOPD II (P1 and P2) (fig. S1) (6). MOPD II patients do not show an enhanced predisposition to cancer; consistent with this, patient lymphocytes did not show an increased frequency of sister chromatid exchange (table S1), as would be indicative of a defect in DNA repair, and typical of another syndrome associated with significant short stature, namely, Bloom syndrome (MIM 210900).

Fig. 1.

Phenotype of MOPD II patients. (A) P18 at age 8 years 3 months with a height of 84 cm corresponding to a normal size for a female infant aged 1 year 3 months; (B and E) P1 at age 8 years 8 months with a height of 85 cm; (C and F) P2 at age 12 years 6 months with a height of 95 cm and at age 14 years with a height of 96 cm (D) corresponding to a normal size for a female aged 3 years. Note short lower arms especially in P18, mild truncal obesity and premature puberty in P1, significant facial asymmetry in P2 (D), and absence of a sloping forehead typical of microcephaly syndromes. All three patients demonstrate a long nose with prominent tip and hypoplastic alae and small mandible described as typical for patients with MOPD II. (G and H) X-ray and an image of the dorsum of the left hand of patient P2 showing generalized brachydactyly with diaphyseal constriction (overmodeling) of metacarpals and phalanges, as well as abnormal flat shape of the distal radius and ulna epiphyses. (I and J) Hypoplasia and partial agenesis of teeth from patient P2, enamel hypoplasia in teeth from patient P18.

Consanguinity in the respective parents of the two unrelated female patients P1 and P2 presented the possibility of locating a MOPD II locus by homozygosity mapping (6, 7) (Fig. 2A). This approach allows the identification of an autosomal recessive disease locus by tracking its segregation within a common chromosomal segment that originates from a shared recent ancestor and is transmitted through both parents. Genome-wide linkage analysis using polymorphic short tandem repeat markers revealed a single disease locus on chromosome 21q22.3. When a third consanguineous family was included, a maximum lod (logarithm of the odds ratio for linkage) score of 3.7 was obtained at marker D21S1446 (Fig. 2 and fig. S2), confirming linkage to this locus. The linked region encompasses 4.6 megabases at the distal end of chromosome 21 and contains the pericentrin (PCNT) gene, which we considered a suitable candidate gene because of its postulated role in chromosome segregation. Mutational analysis of the 47 exons of PCNT in 25 unrelated patients with a clinical diagnosis of MOPD II, including those from the three linked families, revealed homozygous and compound heterozygous null mutations distributed throughout the gene in all patients (table S2 and Fig. 2A). We observed a total of 29 different mutations consisting of 12 stop mutations and 17 frameshift mutations (4 splice-site mutations, 2 small insertions, 10 small deletions, 1 exon deletion). Two mutations occurred twice in unrelated patients, namely, R1923X, in patients P3 and P4, and c.841_842insG in patients P11 and P13. R1923X occurred independently twice, as the respective PCNT haplotypes differed in a total of 24 single-nucleotide polymorphisms, whereas c.841_842insG appears to have been transmitted through an unknown common ancestor in patients P11 and P13 (both of Turkish origin), because these patients were identical for all polymorphic sites identified within the PCNT genomic region. In contrast, 17 patients with a clinical diagnosis of MOPD I or III, Seckel syndrome, or unclassified growth retardation syndromes showed no PCNT mutations. Absence of the PCNT protein (also known as kendrin, PCNT2, or PCNTB) was confirmed by Western blot analysis of lymphoblastoid cell lines from patients P18 and P6 (Fig. 2B). It is noteworthy that both investigated heterozygous parents of patient 18 showed reduced protein levels in lymphoblasts. This might explain our finding of significant reduction of the mean height of heterozygous MOPD II parents (table S3). PCNT is apparently not sensitive to gene dosage alterations, because mRNA levels were normal in patients with either monosomy or trisomy of the PCNT locus (fig. S3A). MOPD II patients showed either normal or variably diminished mRNA levels (fig. S3B), most likely due to varying degrees of nonsense-mediated mRNA decay resulting from pretranslational mRNA surveillance mechanisms (8). Our findings thus characterize MOPD II as a distinct clinical entity caused by biallelic loss-of-function mutations in PCNT. Given that all PCNT mutations observed in MOPD II patients are mutations leading to a loss of functional protein, it remains to be determined whether PCNT missense variants are associated with incomplete or distinct phenotypes.

Fig. 2.

Pedigrees used for linkage analysis and the respective homozygous mutations identified in PCNT. (A) Families 1 and 2 were used for the whole-genome scan; families 1, 2, and 15 were used for fine mapping. Individuals marked with asterisks were included in the linkage analysis. (B) Western blot analysis of lymphoblastoid cell lines from MOPD II patients P18 and P6, the parents of P18 (F18, father, and M18, mother), and normal controls (C1 to C3). Note the undetectable PCNT (370 kD) in P18 and P6 and reduction of protein level in both parents. *SeeBluePlus2 Prestained standard (Invitrogen, Carlsbad, CA).

PCNT is a giant coiled-coil protein (∼370 kD) that localizes specifically to centrosomes throughout the cell cycle (9). The centrosome is a cell component that organizes cytoplasmic organelles and primary cilia in interphase cells, and mitotic spindle microtubules to ensure proper chromosome segregation during cell division (10). PCNT and AKAP9 (A kinase anchor protein 9; formerly known as CG-NAP) share a highly related C-terminal calmodulin-binding domain and mediate, in a noncompensating manner, nucleation of microtubules by anchoring the γ-tubulin ring complex, which initiates the assembly of the mitotic spindle apparatus (9, 11, 12). Pericentrin and AKAP9 are orthologs of the yeast Spc110 protein, whose absence causes defective spindle formation and results in a lethal failure to segregate chromosomes in the budding yeast (13, 14). Programmed cell death (apoptosis) after activation of mitotic checkpoints and arrest of cells in G2 phase–to-mitosis transition was shown in some, but not all, vertebrate cell lines depleted of PCNT by small interfering RNA (12). It is likely that pericentrin-depleted human cells are more susceptible to death because of defective mitosis and chromosome segregation. This would result in a decrease in total cellularity of the embryo and growth restriction in the adult. In accord with this hypothesis, we observed abnormal mitotic morphology in 71% of MOPD II fibroblast cells (Fig. 3), together with low-level mosaic variegated aneuploidy (MVA) and premature sister chromatid separation (PCS) (fig. S4 and table S4). As suggested for the centrosome in general, our findings would indicate an additional role of PCNT in the spindle assembly checkpoint, in the absence of which cells do not arrest in metaphase but prematurely separate sister chromatids and then exit mitosis (15). PCS and MVA, at higher rates than we observed in MOPD II cells, are characteristic of individuals with MVA syndrome (MIM 257300) characterized by cancer susceptibility, growth retardation of intrauterine onset, and microcephaly because of homozygous mutations in the gene encoding BUBR1, a protein, which is known to be involved in the mitotic spindle checkpoint and the initiation of apoptosis in polyploidy cells (16).

Fig. 3.

Abnormal mitotic morphology of patient fibroblasts. Immunofluorescence images of fibroblast cells with antibodies against PCNT (red) and α-tubulin (green), and 4′,6′-diamidino-2-phenylindole (DAPI) staining of chromosomes (blue). (A to D) Representative morphology of fibroblasts from a healthy individual during (A) interphase, (B) metaphase, (C) anaphase, and (D) cytokinesis. (E to L) Undetectable PCNT in fibroblasts from the MOPD II patient P1 in interphase (E) and during mitosis [(F) to (L)] as well as representative examples of abnormal morphology with disorganized mitotic microtubules during prometaphase (I), metaphase [(F) and (J)] and anaphase (G); incorrect vertical orientation of metaphases (J); and disorganized cytokinesis [(H), (K), and (L)] with abnormal nuclei pattern (K). Clearly abnormal spindle pattern was observed in 71% of mitotic fibroblasts from the MOPD II patient (n = 100; control 9%, n = 100; P < 3 × 10–20; Fisher's exact test).

Although the precise pathogenic mechanisms involved remain unclear, it is noteworthy that mutations in centrosomal and mitotic spindle–related genes have now been identified in three forms of primary microcephaly (CDK5RAP2: MCPH3, MIM 604804; ASPM: MCPH5, MIM 608716; CENPJ: MCPH6, MIM 608393). In addition, biallelic mutations in MCPH1, which functions in the regulation of chromosome condensation, have been reported in primary microcephaly with mental retardation and short stature (MIM 606858).

There is an ongoing debate as to whether the Late Pleistocene hominid fossils from the island of Flores, Indonesia, represent a diminutive, small-brained new species, Homo floresiensis, orpathological modern humans (1728). We note that individuals with MOPD II have several features in common with Homo floresiensis, including an adult height of 100 cm, grossly normal intelligence despite severely restricted brain size, absence of a sloping microcephalic morphology, and a number of minor morphological features including facial asymmetry, small chin, abnormal teeth, and subtle bony anomalies of the hand and wrist. Given these similarities, it is tempting to hypothesize that the Indonesian diminutive hominids were in fact humans with MOPD II. With the identification of the genetic basis of MOPD II, this hypothesis may soon be testable.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1151174/DC1

Materials and Methods

Figs. S1 to S4

Tables S1 to S4

References

References and Notes

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