To understand the role of 5'UTR of ENG we sequenced the noncoding region of exon 1 of ENG for 154 unrelated patients/probands with 2 or more clinical diagnostic criteria. These results revealed three sequence changes; c.-127C > T, c.-205A > C, and c.-9G > A. Out of 154, three had c.-127C > T, one had c.-205A > C, and three had c.-9G > A mutation. When available, probands' family members were studied to see if the mutation tracked with the disease. For the c.-127C > T mutation, we included a family from Northern Spain to provide additional information about this mutation. Clinical manifestations of the probands and their evaluated family members are summarized in Table . Cases listed as having no solid organ involvement had screening for pulmonary AVMs (PAVMs) by contrast echocardiogram and/or chest computed tomography (CT) and for brain AVMs by a contrasted magnetic resonance imaging (MRI), and physical examination and medical history that did not suggest other AVMs. Sequencing results of 134 healthy control samples did not reveal any sequence change in the 5'UTR of ENG.
Molecular results and clinical findings in probands and family members
The c.-127C > T heterozygous change was found in three out of the 154 clincal HHT cases and in one case from Northern Spain (family 4). For one of these cases (family 1), an affected sibling was also available and was found to be positive for the mutation. Two of the siblings met clinical diagnostic criteria with frequent epistaxis, typical telagiectasia, PAVMs, and gastrointestinal (GI) telangiectasia. The second patient (proband 2) was a member of a family (family 2) linked to the ENG by locus specific short tandem repeat (STR) markers. The ACVRL1 region was excluded in this family (data not shown). There were 5 clinically affected family members available for the family segregation study (Figure ). The mutation is carried by all studied family members affected with HHT. Although our 67 year old proband did not have any solid organ involvement, an 18 year old grandniece had a spinal AVM, an 8 year old grandnephew had cerebral AVMs (CAVMs), and three other affected family members had pulmonary AVMs (PAVMs). Family members of the third patient (proband 3) were not available for family segregation study. Proband 3 had PAVMs, telangiectases on the face and 2-6 episodes/week of epistaxis. The c.-127C > T mutation was also found in one family proband from Northern Spain (family 4). No other affected family member, including his deceased mother, was available for a segregation study. But the mutation was not seen in his unaffected father or brother (Figure ).
Figure 1 A. Family segregation study for family 2. The pedigree for family 2 is shown. The c.-127C > T mutation was shown to segregate among affected individuals in this family, where 5 clinically affected family members were available for the family segregation (more ...)
The sequence ideogram and neighboring sequences of the c.-127C > T mutation are shown in Figure . This sequence alteration creates a potential AUG initiation codon at base -127 from translation initiation of the ENG
gene. The c.-127C > T change is not reported in the NCBI dbSNP database. NetStart 1.0 Prediction Program [25
] predicts that this mutation creates a new translation start site (TIS) with an altered reading frame (Figures and ). Interestingly, the sequence surrounding the new TIS fits well with the Kozak consensus and other motifs that play a major role in the initiation of the translation process [26
], suggesting that this new TIS may be functionally active. Because translation usually initiates solely at the first ATG codon in an adequate context, it is likely that the new TIS at -126/-128 is competing advantageously with the constitutive TIS at +1. To test this hypothesis, we generated a mutant construct in a full length endoglin cDNA, that contains the 5'UTR [23
], where the c.-127C > T change was introduced (Figure ). The wild type and mutant constructs were cloned into an expression vector and the levels of endoglin protein expression were assessed by transient transfection in the monkey cell line COS-7. As shown in Figure , we found that protein expression levels of the mutant endoglin construct c.-127C > T were markedly reduced (74%) with respect to the wild type construct. This result suggests that the c.-127C > T mutation generates a functional TIS out of frame that interferes with translation initiation of the constitutive ATG at +1, leading to endoglin haploinsufficiency.
Figure 2 A. Sequencing results from one individual with the c.-127C > T heterozygous mutation. The forward sequence is shown. The arrow indicates the position of the mutation. 2B. Sequencing results from two individuals (one homozygous and one heterozygous) (more ...)
Figure 3 Schematic representation of wild type and mutant versions of endoglin. The 5'UTR, the 3'UTR and the region corresponding to the ORF are indicated (top). The sequence of wild type (WT) and mutants (c.-127C > T; c.-9G > A; c.-9G > (more ...)
Figure 4 Functional analysis of ATG endoglin mutants. COS-7 cells were transfected with wild type (WT) or endoglin mutants 4A: c.-9G > A (-9), c.-9G > A & c.+1A > G (-9&+1) or c.-127C > T (-127), and 4B: c.-205A (more ...)
The c.-9G > A mutation (Figure ) was found in three HHT families. Three family members were available from family 6 (Figure ). All carried the mutation and had infrequent epistaxis and telangiectases with no solid organ involvement. Proband 7 was 27 years old when she was examined. She had a few telangiectases on her face and infrequent epistaxis only during her childhood. She did not have any solid organ involvement. There were no other family members available for this study. All of these patient's clinical findings were relatively mild, none of them had solid organ involvement or severe/frequent epistaxis causing anemia or blood transfusion.
Proband 8 was found to be homozygous for c.-9G > A mutation (Figure ). In order to confirm this result and to rule out primer binding site polymorphisms, this region was sequenced with three different primer sets. Multiplex ligation dependent probe amplification (MLPA) method was used to test for a large deletion of the region. MLPA results and sequencing with different primer sets confirmed that proband 8 carries two copies of the mutant allele for this region (data not shown).
Proband 8 had daily epistaxis and telangiectases on his lips, tongue, ear, hands, face and pharynx. His son's clinical findings were not as profound as his father. He had a few telangiectases on his lips and face, and epistaxis. Neither of them had solid organ involvement. His son was not available for molecular testing; however, he is an obligate carrier for the mutation as his father is homozygous. The parents of the proband were deceased, thus not available for examination or testing. Neither was known by the proband to have nosebleeds or telangiectasia. The mother reportedly died at age 42 of tuberculosis and father of a myocardial infarct in the decade of his 60s.
The c.-9G > A mutation is not reported in NCBI dbSNP database, nor it was seen in our control group. This mutation is predicted to create a new TIS with the same reading frame as endoglin and a resulting protein which contains three additional amino acids in the leader sequence (Figure ). Of note, the length of the resulting leader sequence (28 amino acids) is within the normal size range of cleavable amino terminal signal peptides in precursor proteins [28
]. To assess whether the new TIS at -9 was affecting the expression of the endoglin protein, we generated two different mutant constructs in a full length endoglin cDNA that contains the 5'UTR. A single mutant contained the c.-9G > A change of the HHT patients, while a double mutant contained both the c.-9G > A mutation plus the c.1A > G mutation. Functional studies of the c.-9G > A mutation showed that expression of the mutant protein was reduced approximately 20% compared to the wild type protein (Figure ). To prove that the ATG at -9 was functioning as a real TIS, the constitutive initiation site at position 1 was abolished in the double mutant (c.-9G > A and c.1A > G). Thus, transfection studies with this double mutant demonstrated that the corresponding endoglin protein was expressed, although at a much lower level (60%), as compared to 81% with the c.-9G > A construct. While the predicted mature endoglin protein driven by the TIS at -9 is identical to the one driven by the constitutive TIS at +1, the decreased expression levels of endoglin with the c.-9G > A mutation are likely due to a lower translation efficiency and/or a less efficient processing of the endoglin precursor protein through the secretory pathway. Taken together, these results suggest that the mutation c.-9G > A confers slightly reduced expression of the mutant protein that is compatible with the mild effect in heterozygosis and a more severe, but still classical, HHT phenotype in homozygosis.
The c.-205A > C heterozygous variant was found in one patient (Proband 5). Three family members were available for study. It was not found in a brother and father with infrequent, recurring nosebleeds and telangiectases in characteristic locations, but was identified in an asymptomatic mother. This variant was not found in dbSNP database, nor was it found in healthy controls. However, based on in silico analyses this variant is not predicted to have a significant effect on the regulation of the translation or transcription. In the family segregation study this mutation does not track with symptoms of HHT, and in silico analysis does not support pathogenicity. Expression analysis of the mutant construct (c.-205A > C) showed similar protein levels as the wild type construct (Figure ) confirming that it is a benign sequence change.