These authors contributed equally to this work. AUTHOR CONTRIBUTIONS The project was conceived and the experiments were planned by E.K. and B.W. We would like to note that Y.A. and K.E.B. had a comparable contribution to this study. The review of phenotypes and the sample collection were performed by B

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.

In a consanguineous Turkish family, a locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI) was mapped to chromosome 2q31.1-2q33.1. Microsatellite marker analysis in the complete family determined the critical linkage interval that overlapped with DFNB27, for which the causative gene has not yet been identified, and DFNB59, a recently described auditory neuropathy caused by missense mutations in the DFNB59 gene. The 352-amino acid (aa) DFNB59 gene product pejvakin is present in hair cells, supporting cells, spiral ganglion cells, and the first three relays of the afferent auditory pathway. A novel homozygous nonsense mutation (c.499C>T; p.R167X) was detected in the DFNB59 gene, segregating with the deafness in the family. The mRNA derived from the mutant allele was found not to be degraded in lymphocytes, indicating that a truncated pejvakin protein of 166 aa may be present in the affected individuals. Screening of 67 index patients from additional consanguineous Turkish families with autosomal recessive hearing impairment revealed a homozygous missense mutation (c.547C>T; p.R183W) that segregates with the hearing impairment in one family. Furthermore, in a panel of 83 Dutch patients, two additional novel mutations (c.509_512delCACT; p.S170CfsX35 and c.731T>G; p.L244R), which were not present in ethnically matched controls, were found heterozygously. Together, our data indicate that also nonsense mutations in DFNB59 cause nonsyndromic hearing loss, but that mutations in DFNB59 are not a major cause of nonsyndromic hearing impairment in the Turkish and Dutch population.

In conclusion, novel ALDH1A3 mutations identified in the present study confirm the pivotal role of ALDH1A3 in human eye development. Autistic features, previously reported as an associated finding, were considered to be the result of social deprivation and inadequate parenting during early infancy in the presented families.

Microcephalic primordial dwarfism (MPD) is a group of autosomal recessive inherited single-gene disorders with intrauterine and postnatal global growth failure. Seckel syndrome is the most common form of the MPD. Ten genes are known with Seckel syndrome. Using genome-wide SNP genotyping and homozygosity mapping we mapped a Seckel syndrome gene to chromosomal region 4q28.1-q28.3 in a Turkish family. Direct sequencing of PLK4 (polo-like kinase 4) revealed a homozygous splicing acceptor site transition (c.31-3 A>G) that results in a premature translation termination (p.[=,Asp11Profs*14]) causing deletion of all known functional domains of the protein. PLK4 is a master regulator of centriole biogenesis and its deficiency has recently been associated with Seckel syndrome. However, the role of PLK4 in genomic stability and the DNA damage response is unclear. Evaluation of the PLK4-Seckel fibroblasts obtained from patient revealed the expected impaired centriole biogenesis, disrupted mitotic morphology, G/M delay, and extended cell doubling time. Analysis of the PLK4-Seckel cells indicated that PLK4 is also essential for genomic stability and DNA damage response. These findings provide mechanistic insight into the pathogenesis of the severe growth failure associated with PLK4-deficiency.