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Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity., This work was supported by a grant from the Spanish Ministry of Science and Innovation (PID2019-105620RB-I00/AEI/10.13039/501100011033).

Resumen: Introducción: La enfermedad de Hirschsprung está causada por un defecto de la migración celular desde la cresta neural hasta el tracto gastrointestinal, resultando en la ausencia de neuronas en el plexo mientérico. Mutaciones en varios genes han sido asociadas a la enfermedad de Hirschsprung, la mayoría afectando a la vía del protooncogén RET. El objetivo de este estudio es la descripción de mutaciones tanto descritas como nuevas asociadas a la enfermedad de Hirschsprung, así como sus implicaciones pronósticas. Material y métodos: Análisis retrospectivo de pacientes con enfermedad de Hirschsprung y resultados genéticos positivos desde 1970 hasta 2013. Resultados: En la serie global, 21 pacientes tenían resultados genéticos positivos, 17 de ellos afectando la vía del protooncogén RET. Dos de las mutaciones son nuevas y no han sido previamente descritas en la literatura médica. Conclusiones: El protooncogén RET es el principal gen asociado a la enfermedad de Hirschsprung. Todavía hay múltiples mutaciones desconocidas relacionadas con la patogenia de la enfermedad. El estudio genético del gen RET debe formar parte del estudio diagnóstico de todos los pacientes con enfermedad de Hirschsprung, así como de sus familiares de primer grado en caso de que las mutaciones estén asociadas a los síndromes MEN2A y MEN2B. Abstract: Introduction: Hirschsprung disease is caused by an impairment in cell migration from the neural crest to the gastrointestinal tract, resulting in an absence of neurons in the myenteric plexus. Many mutations in several genes have been associated to Hirschsprung disease; most of them affecting the RET proto-oncogen pathway. The purpose of this study is the description of novel and known mutations in genes associated to Hirschsprung disease and their prognostic implications. Material and methods: Retrospective analysis of patients with Hirschsprung disease and positive genetic studies evaluated from 1970 to 2013. Results: We found 21 positive genetic studies in the global series, 17 of them involving the RET proto-oncogene. Two of the mutations are novel and they have not been reported in the medical literature. Conclusions: The RET protooncogene is the main gene associated with Hirschsprung disease. There are still multiple unknown mutations related to the pathogenesis of the disease. The study of this gene must be part of the work-up of all patients with Hirschsprung disease, as well as their first degree relatives if the mutation is associated with MEN2A and MEN2B syndromes.

[Purpose]: This study aimed to identify the genetic cause of a new multiple congenital anomalies syndrome observed in three individuals from two unrelated families., [Methods]: Clinical assessment was conducted prenatally and at different postnatal stages. Genetic studies included exome sequencing (ES) combined with single-nucleotide polymorphism (SNP) array based homozygosity mapping and trio ES. Dermal fibroblasts were used for functional assays., [Results]: A clinically recognizable syndrome characterized by severe developmental delay, variable brain anomalies, congenital heart defects, dysmorphic facial features, and a distinctive type of synpolydactyly with an additional hypoplastic digit between the fourth and fifth digits of hands and/or feet was identified. Additional features included eye abnormalities, hearing impairment, and electroencephalogram anomalies. ES detected different homozygous truncating variants in MAPKAPK5 in both families. Patient-derived cells showed no expression of MAPKAPK5 protein isoforms and reduced levels of the MAPKAPK5-interacting protein ERK3. F-actin recovery after latrunculin B treatment was found to be less efficient in patient-derived fibroblasts than in control cells, supporting a role of MAPKAPK5 in F-actin polymerization., [Conclusion]: Our data indicate that loss-of-function variants in MAPKAPK5 result in a severe developmental disorder and reveal a major role of this gene in human brain, heart, and limb development., We are grateful to patients and their parents for their participation in this study. The work at IIB was financially supported by the Spanish Ministry of Science, Innovation and Universities (PID2019-105620RB-I00/AEI/10.13039/501100011033 and SAF2016‐75434‐R (AEI/FEDER, UE).

Glucogenosis type IX is caused by pathogenic variants of the PHKA2 gene. Herein, we report a patient with clinical symptoms compatible with Glycogen Storage Disease type IXa. PYGL, PHKA1, PHKA2, PHKB and PHKG2 genes were analyzed by Next Generation Sequencing (NGS). We identified the previously undescribed hemizygous missense variant NM_000292.2(PHKA2):c.1963G > A, p.(Glu655Lys) in PHKA2 exon 18. In silico analyses showed two possible pathogenic consequences: it affects a highly conserved amino acid and disrupts the exon 18 canonical splice donor site. The variant was found as a “de novo” event.

[Background]: Low serum levels of alkaline phosphatase (ALP) are a hallmark of hypophosphatasia. However, the clinical significance and the underlying genetics of low ALP in unselected populations are unclear. [Methods]: In order to clarify this issue, we performed a clinical, biochemical and genetic study of 42 individuals (age range 20-77 yr) with unexplained low ALP levels. [Results]: Nine had mild hyperphosphatemia and three had mild hypercalcemia. ALP levels were inversely correlated with serum calcium (r = - 0.38, p = 0.012), pyridoxal phosphate (PLP; r = - 0.51, p = 0.001) and urine phosphoethanolamine (PEA; r = - 0.49, p = 0.001). Although many subjects experienced minor complaints, such as mild musculoskeletal pain, none had major health problems. Mutations in ALPL were found in 21 subjects (50%), including six novel mutations. All but one, were heterozygous mutations. Missense mutations were the most common (present in 18 subjects; 86%) and the majority were predicted to have a damaging effect on protein activity. The presence of a mutated allele was associated with tooth loss (48% versus 12%; p = 0.04), slightly lower levels of serum ALP (p = 0.002), higher levels of PLP (p < 0.0001) and PEA (p < 0.0001), as well as mildly increased serum phosphate (p = 0.03). Ten individuals (24%) had PLP levels above the reference range; all carried a mutated allele. [Conclusion]: One-half of adult individuals with unexplained low serum ALP carried an ALPL mutation. Although the associated clinical manifestations are usually mild, in approximately 50% of the cases, enzyme activity is low enough to cause substrate accumulation and may predispose to defects in calcified tissues., This work was supported by an unrestricted grant from Alexion.

El síndrome de Crouzon es una craneosinostosis com-pleja debida a mutaciones en el receptor del factor de crecimiento fibroblástico (FGFR) tipo 2. Se presenta una paciente de sexo femenino con la asociación de síndrome de Crouzon más acantosis nigricans (CAN). El estudio molecular mostró una mutación puntual (Ala391Glu) en el dominio transmembrana de otro FGFR, el tipo 3 (FGFR3) (a escasas bases de distancia de la mutación más frecuente de acondroplasia, la Gly380Arg).El síndrome de CAN es una entidad recientemente des-crita, cuya clínica y alteración molecular es diferente a la del síndrome de Crouzon. Estas diferencias son importantes cuando es necesario establecer una estrategia para realizar estudios moleculares y para el diagnóstico prenatal de esta entidad. : Crouzon's syndrome is a complex craniosynostosis disorder due to mutations in fibroblast growth factor receptor (FGFR) type 2.We report a female patient with Crouzon's syndrome associated with acanthosis nigricans. The molecular abnormality in this patient is a point mutation (Ala391Glu) in the transmembrane domain of another FGFR (type 3), which is very close to the mutation (Gly380Arg) most frequently observed in achondroplasia.Acanthosis nigricans is an emerging disorder. Its clinical features and molecular findings differ from those of isolated Crouzon's syndrome. These data are very useful when molecular tests are required for prenatal diagnosis.

Osteogenesis imperfecta or >brittle bone disease> is a collagen type I-related condition associated with osteoporosis and increased risk of bone fractures. Using a combination of homozygosity mapping and candidate gene approach we identified a homozygous single basepair deletion (c.1052delA) in SP7/Osterix (OSX) in a consanguineous patient with recessive osteogenesis imperfecta. The clinical findings of this patient include recurrent fractures, mild bone deformities, delayed tooth eruption, normal hearing, and white sclera. OSX encodes a transcription factor containing three Cys2-His2 zinc-finger DNA-binding domains at its C-terminus, which in mice has been shown to be essential for bone formation. The frameshift caused by the c.1052delA deletion removes the last 81 amino acids of the protein including the third zinc-finger motif. This finding adds a new locus to the spectrum of genes associated with osteogenesis imperfecta and reveals that SP7/OSX also plays a key role in human bone development.

Mutations in BMP1 cause osteogenesis imperfect (OI) type XIII (MIM 614856), a probably not so uncommon form of autosomal recessive OI. (Editor's Note: See Chapters 1 and 2 for discussion of OI types related to specific mutations.) We have studied an Egyptian consanguineous family with two children initially diagnosed as OI type III. They had blue sclera, severe kyphoscoliosis and large umbilical hernia. Genetic analysis of this family performed by homozygosity mapping showed lack of linkage to any of the previously known AR-OI loci but identified a homozygous 10.27. Mb DNA segment on chromosome 8p in both affected children comprising the type I collagen C-propeptide protease gene BMP1. Direct sequencing of BMP1 in the proband revealed a Phe249Leu homozygous missense change within the BMP1/mTLD astacin protease domain. Phe249 is a residue which is invariable in all invertebrate and vertebrate members of the astacin family of metalloproteases. A few months later, using a combination of whole-exome sequencing and filtering for homozygous stretches of identified variants, another mutation was reported by other authors in two affected sibs of a consanguineous family from Turkey. They showed increased bone mineral density and multiple recurrent fractures. The mutation in BMP1, a 34. G>C transversion in exon 1 resulting in a Gly12Arg (G12R) substitution within the signal peptide, impaired protein's localization to the endoplasmic reticulum, the correct post-translational glycosylation and its secretion. BMP1 expands the number of growing genes involved in AR-OI and demonstrates the high genetic complexity of the collagen I pathway disorders.