RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

A novel mutation in CDK5RAP2 gene causes primary microcephaly with speech impairment and sparse eyebrows in a consanguineous Pakistani family.

CDK5RAP2 gene encodes a centrosomal protein, highly expressed in fetal brain and essentially indispensable for its normal development, as biallelic mutations in it lead to primary microcephaly (MCPH). Despite being known as MCPH linked gene for more than a decade, the phenotypic spectrum of CDK5RAP2 mutations is still under explored as only eleven families have been reported worldwide. Here, we analyzed a consanguineous Pakistani MCPH family, characterized by moderate to severe intellectual disability, speech impairment, moderately short stature and sparse eyebrows. Whole exome sequencing of the proband identified a 2bp duplication in exon 34 of CDK5RAP2 that causes frame-shift, leading to a premature stop codon. The resultant transcript is resistant to nonsense mediated decay, suggesting that the mutation leads to a truncated protein lacking C-terminal domains; CDK5R1, and Cnn motif 2 (CM2), required for its localization to centrosome and Golgi Apparatus. Clinical variability observed in the family highlights the importance of further detailed clinical description of patients with CDK5RAP2 mutations.

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family.

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

Association between the development of the body axis and the craniofacial skeleton studied by immunohistochemical analyses using collagen II, Pax9, Pax1, and Noggin antibodies.

STUDY DESIGN: Immunohistochemical analyses on the axial skeleton from wild type mice. OBJECTIVE: In the clinic, we have previously observed cervical spine defects associated with deviations in the posterior part of the occipital bone and with morphologic and functional variations in the craniofacial skeleton. As examples, cervical spine fusions occurred frequently in patients with mandibular overjet and even more frequently and more caudally in the cervical spine in patients with sleep apnoea. The aims of the present study were to elucidate this association between the spine and the cranium by comparing gene expression domains of important developmental genes known to be involved in vertebral column formation with gene expression in the craniofacial region. SUMMARY OF BACKGROUND DATA: This is the first study looking specifically on gene expression in the basilar part of the occipital bone that is formed around the cranial part of the notochord, thus connecting the spine and the craniofacial skeleton. METHODS: The material consisted of 4 mouse embryos p.c. day 13.5, NMRI wild-type mice, from the same litter. The body axis, the cranial base, and the craniofacial area were studied by immunohistochemical analyses using Collagen II, Pax9, Pax1, and Noggin antibodies. RESULTS: Pax1 expression was highly similar in the posterior part of the occipital bone and in the vertebral column, indicating that the basilar part of the occipital bone from a developmental standpoint can be considered the uppermost vertebra. Pax9 and Noggin expression domains were in accordance with those described previously. CONCLUSION: The present study supports that the basilar part of the occipital bone may be regulated by similar developmental mechanisms as the vertebral column and may thus be regarded the uppermost vertebra. Thus, the clinically observed association between the cervical column and the craniofacial area has been proved by immunohistochemical methods.

Genetic heterogeneity in microcornea-cataract: five novel mutations in CRYAA, CRYGD, and GJA8.

PURPOSE: To unravel the molecular genetic background in families with congenital cataract in association with microcornea (CCMC, OMIM 116150). METHODS: CCMC families were recruited from a national database on hereditary eye diseases; DNA was procured from a national gene bank on hereditary eye diseases and by blood sampling from one large family. Genomewide linkage analysis, fine mapping, and direct genomic DNA sequencing of nine cataract candidate genes were applied. Restriction enzyme digests confirmed identified mutations. RESULTS: Analyses of 10 Danish families with hereditary congenital cataract and microcornea revealed five novel mutations. Three of these affected the crystallin, alpha-A gene (CRYAA), including two mutations (R12C and R21W) in the crystallin domain and one mutation (R116H) in the small heat shock domain. One mutation (P189L) affected the gap junction protein alpha 8 (GJA8), and one mutation (Y134X) was detected in crystallin gamma-D (CRYGD). CONCLUSIONS: The identification of a CRYGD mutation adds another gene to those that may be mutated in CCMC and underscores the genetic heterogeneity of this condition. Three CRYAA mutations at the R116 position, in association with CCMC, suggest that R116 represents a CCMC-mutational hotspot. The CCMC phenotype demonstrates variable expression with regard to cataract morphology and age of appearance. Clinical heterogeneity, including additional malformation of the anterior segment of the eye, confirm that dedicated cataract genes may be involved in the largely unknown developmental molecular mechanisms involved in lens-anterior segment interactions.

Branchio-oto-renal syndrome: detection of EYA1 and SIX1 mutations in five out of six Danish families by combining linkage, MLPA and sequencing analyses.

The branchio-oto-renal (BOR) syndrome is an autosomal-dominant disorder characterized by hearing loss, branchial and renal anomalies. BOR is genetically heterogeneous and caused by mutations in EYA1 (8q13.3), SIX1 (14q23.1), SIX5 (19q13.3) and in an unidentified gene on 1q31. We examined six Danish families with BOR syndrome by assessing linkage to BOR loci, by performing EYA1 multiplex ligation-dependent probe amplification (MLPA) analysis for deletions and duplications and by sequencing of EYA1, SIX1 and SIX5. We identified four EYA1 mutations (c.920delG, IVS10-1G>A, IVS12+4A>G and p.Y591X) and one SIX1 mutation (p.W122R), providing a molecular diagnosis in five out of the six families (83%). The present, yet preliminary, observation that renal and temporal bone malformations are less frequent in SIX1-related disease suggests a slightly different clinical profile compared to EYA1-related disease. Unidentified mutations impairing mRNA expression or further genetic heterogeneity may explain the lack of mutation finding in one family despite LOD score indications of EYA1 involvement.

The congenital "ant-egg" cataract phenotype is caused by a missense mutation in connexin46.

PURPOSE: "Ant-egg" cataract is a rare, distinct variety of congenital/infantile cataract that was reported in a large Danish family in 1967. This cataract phenotype is characterized by ant-egg-like bodies embedded in the lens in a laminar configuration and is inherited as an autosomal dominant trait. We retrieved the family and performed linkage analysis to determine the disease locus and identify the mutated gene. METHODS: The family (CC00103) was identified in a National Register of Hereditary Eye Diseases and updated based on The Danish Civil Register System. Genome wide linkage analysis and haplotyping using STS marker systems were carried out to achieve a LOD score above 3. The disease-causing candidate gene was sequenced and the mutation was identified and verified by restriction enzyme digestion of genomic DNA from all individuals in family CC00103 and 60 healthy controls. RESULTS: Linkage analysis resulted in a LOD score of 3.91 for marker D13S1275 located close to the known cataract gene GJA3. A novel missense mutation c.32T > C (L11S), was found by sequencing DNA from two affected members. The mutation was present in all affected individuals and was neither found in unaffected family members nor in 60 healthy individuals by restriction enzyme digests. CONCLUSIONS: The congenital "ant-egg" cataract phenotype is caused by a L11S mutation in connexin46 (Cx46) located in the signal peptide domain. Further studies are needed to unravel the mechanism leading to the formation of the "ant-eggs".

Mapping of hereditary trichilemmal cyst (TRICY1) to chromosome 3p24-p21.2 and exclusion of beta-CATENIN and MLH1.

Trichilemmal cysts (also named pilar cyst) derived from the outer root sheath of the deeper parts of the hair follicle can segregate dominantly, and are caused by a yet unknown gene. In order to identify candidate genes for this trait we have ascertained a Danish family with 38 persons (11 affected), and carried out a genome wide scan with 580 DNA micro-satellite markers to identify the locus for a gene, which we termed TRICY1 (for trichilemmal cysts). We found tight linkage to D3S1277 (Z = 4.63; theta(M = F) = 0.00), with flanking markers D3S2432 (Z = 1.59; theta(M = F) = 0.08), and D3S3685 (Z = 2.69; theta(M = F) = 0.08) spanning 10.3 Mb on chromosome 3p24-p21.2. We sequenced two candidate genes previously reported in inherited hair defects, CTNNB1 and MLH1 but failed to detect mutations in exons and intron-exon bounders.

HOXD13 polyalanine tract expansion in classical synpolydactyly type Vordingborg.

In 1927, Oluf Thomsen, in a classic paper, described a seven-generation family with autosomal dominant axial synpolydactyly (SPD)--the Vordingborgtyp of axis duplication and dysostosis. Expansion of a polyalanine tract in the HOXD13 gene is known to cause synpolydactyly. We have rediscovered part of the family described by Thomsen, and detected a 9 triplet polyalanine expansion within HOXD13segregating with the disorder. The phenotypic spectrum in mutation carriers ranged from severe to inapparent bone malformations. In the latter case, only dermatoglyphics revealed the genetic status.