Functional and in silico assessment of MAX variants of unknown significance.

UNLABELLED: The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC's E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC's ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX. KEY MESSAGES: A functional assay assesses the effects of MAX VUS over MYC transcriptional activity. A consensus computational prediction and the functional assay show high concordance. Variant carriers' clinical and molecular data support the functional assessment.

An unusual phenotype of X-linked developmental delay and extreme behavioral difficulties associated with a mutation in the EBP gene.

We report on a family in which four males over three generations are affected with X-linked recessive developmental delay, learning difficulties, severe behavioral difficulties and mild dysmorphic features. Plasma sterol analysis in three of the four affected males demonstrated increased concentrations of 8-dehydrocholesterol (8-DHC) and cholest-8(9)-enol. All four affected males had a novel hemizygous missense mutation, p.W47R (c.139T>C), in EBP. Functional studies showed raised levels of cholest-8(9)-enol in patient's cultured fibroblast cells, which were suppressed when the cells were incubated with simvastatin. EBP encodes 3ß-hydroxysteroid-delta8, delta7-isomerase, a key enzyme involved in the cholesterol biosynthesis pathway. Mutations in EBP have previously been associated with Conradi-Hunermann-Happle syndrome (CHH), an X-linked dominant disorder characterized by skeletal dysplasia, skin, and ocular abnormalities, which is usually lethal in males. Four previous reports describe X-linked recessive multiple anomaly syndromes associated with non-mosaic EBP mutations in males, two at the same amino acid position, p.W47C. This phenotype has previously been described as "MEND" syndrome (male EBP disorder with neurological defects). The family reported herein represent either a novel phenotype, or an expansion of the MEND phenotype, characterized by extreme behavioral difficulties and a scarcity of structural anomalies. Simvastatin therapy is being evaluated in two males from this family.

A prospective study of brachytelephalangic chondrodysplasia punctata: identification of arylsulfatase E mutations, functional analysis of novel missense alleles, and determination of potential phenocopies.

PURPOSE: The only known genetic cause of brachytelephalangic chondrodysplasia punctata is X-linked chondrodysplasia punctata 1 (CDPX1), which results from a deficiency of arylsulfatase E (ARSE). Historically, ARSE mutations have been identified in only 50% of male patients, and it was proposed that the remainder might represent phenocopies due to maternal-fetal vitamin K deficiency and maternal autoimmune diseases. METHODS: To further evaluate causes of brachytelephalangic chondrodysplasia punctata, we established a Collaboration Education and Test Translation program for CDPX1 from 2008 to 2010. Of the 29 male probands identified, 17 had ARSE mutations that included 10 novel missense alleles and one single-codon deletion. To determine pathogenicity of these and additional missense alleles, we transiently expressed them in COS cells and measured arylsulfatase E activity using the artificial substrate, 4-methylumbelliferyl sulfate. In addition, clinical data were collected to investigate maternal effects and genotype-phenotype correlations. RESULTS: In this study, 58% of males had ARSE mutations. All mutant alleles had negligible arylsulfatase E activity. There were no obvious genotype-phenotype correlations. Maternal etiologies were not reported in most patients. CONCLUSION: CDPX1 is caused by loss of arylsulfatase E activity. Around 40% of male patients with brachytelephalangic chondrodysplasia punctata do not have detectable ARSE mutations or known maternal etiological factors. Improved understanding of arylsulfatase E function is predicted to illuminate other etiologies for brachytelephalangic chondrodysplasia punctata.

Prenatal testing for a novel EBP missense mutation causing X-linked dominant chondrodysplasia punctata.

OBJECTIVE: To determine the pathogenicity of a novel conserved missense mutation, p.Ser98Phe, in the emopamil binding protein (EBP) gene in order to perform a prenatal diagnostic test for X-linked dominant chondrodysplasia punctata (CDPX2) in a male foetus at 50% risk. METHODS: Family members were tested for p.Ser98Phe using PCR and sequence analysis of leucocyte or buccal cell DNA. Haplotype analysis was employed to identify the grandparental chromosome on which p.Ser98Phe had arisen. In silico protein analyses were used to predict whether p.Ser98Phe significantly altered the EBP protein structure. RESULTS: The mutation was detected in the proband and her affected mother but not in the maternal grandmother, who was thought to be mildly affected. However, haplotype analysis showed that p.Ser98Phe had arisen de novo on the grandpaternal X chromosome. Protein secondary structure predictions suggested that p.Ser98Phe alters the properties of the helix within which it is located. CONCLUSION: We concluded that p.Ser98Phe is likely to be pathogenic and proceeded with prenatal testing. The male foetus had not inherited p.Ser98Phe and his unaffected status was confirmed at birth. This family demonstrates some of the difficulties in interpreting the significance of novel missense mutations, particularly when results are needed urgently for prenatal diagnosis.

A family with a novel TSH receptor activating germline mutation (p.Ala485Val).

Autosomal dominant nonautoimmune hyperthyroidism (ADNAH) is caused by gain of function mutations in the TSH receptor (TSHr) gene and characterized by toxic thyroid hyperplasia with a variable age of onset in the absence of thyroid antibodies and clinical symptoms of autoimmune thyroid disease in at least two generations. We report here a Turkish family with a novel TSHr gene mutation with distinct features all consistent with ADNAH. Thyroid function tests of the proband were as follows: free T3: 13.1 pg/ml (N: 1.8-4.6); free T4: 5.1 ng/dl (N: 0.9-1.7); TSH: 0.01 microIU/ml (N: 0.2-4.2); and TSH receptor antibody: 2 IU/ml (N: 0-10). A heterozygous missense mutation in exon 10 of the TSHr gene (c.1454C>T) resulting in the substitution of valine for alanine at codon 485 (p.Ala485Val) was found in the father and his son and daughter. This mutation had arisen de novo in the father. Functional studies of the novel TSHr germline mutation demonstrated a higher constitutive activation of adenyl cyclase than wild type without any effect on phospholipase C activity. In conclusion, our data indicate that gain of function germline mutations in the TSHr gene should be investigated in families with members suffering from thyrotoxicosis and progressive growth of goiter, but without clinical and biochemical evidence of autoimmune thyroid disease. In addition, patients harboring the same mutation of the TSHr gene may show wide phenotypic variability with respect to the age at onset, and severity of hyperthyroidism and thyroid growth.

Acrogeric Ehlers-Danlos syndrome type IV: report of a new patient with additional findings.

We report a 21 year-old Turkish male with acrogeric Ehlers-Danlos syndrome type IV, a rare autosomal dominant disorder. In addition to the usual findings, the patient also had glaucoma and some unusual skeletal features including impressio digitalis in the skull, prognathism of the lower jaw and maxiller hypoplasia, not previously described as part of acrogeric Ehlers-Danlos syndrome type IV. These features expand the phenotypic spectrum of acrogeric Ehlers-Danlos syndrome type IV.