Clinical, genetic, and molecular characterization of hyperphosphatasia with mental retardation: a case report and literature review

Since establishing the craniomaxillofacial research project at the Lebanese American University, we have examined over 100 patients with orofacial dysmorphic features, including a pair of twins suffering from a severe developmental syndrome. Participants provided written informed consent prior to filling out the questionnaire or giving blood samples. Parents of the minor participants provided a written declaration of consent as they were legally authorized representatives. Research and data collection were carried out in compliance with the Helsinki Declaration, with the approval of the LAU Institutional Review Board and the Committee on Human Subjects in Research (CHSR). De-identified data about the neurological, muscular, epithelial, and connective tissues related development was collected and all medical problems were obtained from medical charts.

ALP levels were measured in all patients. Information including brain imaging, EEG, skin biopsies, echocardiograms, and thyroid profile were also performed. Parents were examined and genomic DNA was extracted from peripheral blood of the patients and their two parents using the standard phenol-chloroform extraction procedure.

Whole exome enrichment was performed on genomic DNA using the SureSelect Human All Exon V6 (Agilent). Whole exome sequencing was performed on an Illumina Hi-Seq 3000 platform using a 150 bp paired-end protocol with an average depth of 60x. Reads were aligned against the human assembly GRCh37 using Burrows-Wheeler Aligner (BWA). Data analysis was performed using SAMtools [16], Picard [5], the Genome Analysis Toolkit (GATK) [18] and Variant Effect Predictor (VEP). Polymorphisms were removed by reference to their population frequencies by searching for such mutations in genetic disease databases including OMIM (http://​omim.​org), HGMD (http://​www.​hgmd.​cf.​ac.​uk/​ac/​index.​php), ClinVar (https:​www.​ncbi.​nlm.​nih.​gov/​clinvar), the database of the 1000 Genomes Project (http://​www.​1000genomes.​org/​), ESP6500 (http://​evs.​gs.​washington.​edu/​EVS/​), and ExAC (http://​exac.​broadinstitute.​org). The effects of mutations on protein function were predicted with the aid of PolyPhen 2 (http://​genetics.​bwh.​harvard.​edu/​pph2/​), SIFT (http://​sift.​jcvi.​org), and CADD (https://​cadd.​gs.​washington.​edu/​). The prioritization of genes was conducted using Pubmatrix (https://​pubmatrix.​irp.​nia.​nih.​gov/​) followed by further assessment in relation to clinical characteristics.

There was a first-degree consanguinity between the parents of the twin pair (Fig. 1). No family history of HPMRS was recorded and both parents reported to be young and healthy at the time of conception. The twins were nine-year-old at the time of data collection. They presented hypotonia with a lack of voluntary coordination of muscle movements prohibiting them from walking, absence of speech, and abnormalities in the eye movement with down-slanting eyelids. They both suffered from growth retardation and epilepsy. No signs of autism or hyperactivity were recorded. Head magnetic resonance imaging showed evidence of 2 × 2 cm area of bleed in the left temporal lobe of both affected individuals with the presence of an abnormal vein. No arterial abnormality was seen. The pattern of gyration and myelination was normal as well as the ventricles. Skin biopsy following the description of skin hyperpigmentation confirmed the diagnosis of incontinentia pigmenti, although the WES data did not show any mutation in IKBKG, previously reported to be linked to the condition [25]. The twins had typical facial features with a wide nasal bridge, tent-shaped lips, and a cleft of the anterior and posterior palate together with typical recurrent ear infections. Gastroscopy in one of the twins, 4CF1, showed the presence of marked esophagitis, large hiatal hernia, petechial gastritis, and minimal nodular duodenitis. In the other twin, 5CF1, cardiac ultrasonography showed a congenital ventricular septal defect with the presence of a heart murmur. He also presented a clinodactyly of the fifth digit (Fig. 1 and Table 1).

The analysis of the whole exome data showed that the patient 5CF1 carried a homozygous frameshift mutation in PGAP3, exon 2, c.203delC (p.C68LfsX88) (Fig. 1). The father (2CF1) was heterozygous for the same mutation. Sanger sequencing was used to confirm the mutation in the two above-studied individuals, and to look for its presence in the mother (3CF1) as well as the other twin (4CF1). Results showed that both parents were carriers for the mutation, and both children were homozygous for the mutation. The presence of an A > G substitution in intron 2, 110 base pairs 3′ of the c.203delC change (genomic position 39,685,888 bp), excluded the hemizygous mutation hypothesis. Although the cytosine on the position 203 has previously been shown to be substituted, the current deletion has not been reported previously in any population. No other relevant nucleotide changes were found. In order to confirm the presence of the HPMRS syndrome, a dosage of the level of ALP was done. The results showed that the ALP level was of 390 U/L and 327 U/L for 4CF1 and 5CF1 respectively.

The mutation resulted in a stop codon replacing a cysteine residue at the 68th amino acid position. Thus, the corresponding protein has only 67 amino acids, compared to 320 amino acids in the wild type protein. Phyre2 suggested the 320 amino-acid normal protein to present seven transmembrane alpha helices spanning amino acids 99 to 296 [13]. However, since the mutated protein has only the first 67 amino acids, no transmembrane helices in this protein were suggested by Phyre2 [13]. This indicates that the protein is no longer capable of getting integrated in the membrane of Golgi apparatus, as the literature states [22]. The templates to which both the normal and the mutated proteins were aligned in the Phyre2 software further support this hypothesis (Fig. 2).