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BackgroundCleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (RUNX2)is currently the only known disease-causing gene for CCD, but several studies have suggested locus heterogeneity.MethodsThe cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level.ResultsIn each subject a heterozygous pathogenic variant inCBFBwas detected, whereas no genomic alteration involvingRUNX2was found. ThreeCBFBvariants (one splice site alteration, one nonsense variant, one 2 bp duplication) were shown to result in a premature stop codon. A large intragenic deletion was found to delete exon 4, without affectingCBFBexpression. The effect of a second splice site variant could not be determined but most likely results in a shortened or absent protein. Affected individuals showed similarities withRUNX2-related CCD, including dental and clavicular abnormalities. Normal stature and neurocognitive problems were however distinguishing features.CBFBencodes the core-binding factor β subunit, which can interact with all RUNX proteins (RUNX1, RUNX2, RUNX3) to form heterodimeric transcription factors. This may explain the phenotypic differences betweenCBFB-related andRUNX2-related CCD.ConclusionWe confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants inCBFBin a cohort of eight individuals with clinical and radiographic features reminiscent of CCD.

Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (The cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level.In each subject a heterozygous pathogenic variant inWe confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants in

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses.