Patient 1 She was born at term to first cousin parents of Pakistani origin. She presented to the dermatologist at the age of 9 months with hyperpigmentation and hypertrichosis, particularly on her back and legs (Fig. 1A). Physical examination showed extensive hyperpigmented areas with dark, long hairs over the buttocks, legs and arms. In addition, she showed limited dysmorphism: epicanthal folds, hypertelorism, widely spaced nipples and lipoatrophy of buttocks. A skin biopsy from the hyperpigmented area of her legs showed non-specific inflammation. Figure 1Open in figure viewerPowerPoint (A) Marked hypertrichosis on the lower legs. (B) Hypermelanosis on the inner aspects of the thighs. (C) Close up of (B) showing the marked hypermelanosis and hypertrichosis. In the subsequent 2 yr, failure to thrive became clear and she suffered from recurrent pyrexia’s and chronic diarrhoea. She was noted to have hepatosplenomegaly, lymphadenopathy, anaemia and mild ascites with peripheral oedema. Numerous investigations failed to explain the underlying cause. At 4 yr of age, because of the polyuria and polydipsia, she was diagnosed with diabetes mellitus necessitating insulin treatment. Islet cell and glutamic acid decarboxylase antibodies were negative. As a result of the persistent diarrhoea, faecal stool elastase was measured, which was found to be undetectable, and pancreatic enzyme replacement therapy was started. Diarrhoea persisted however, and growth slowed down [at 4.4 yr height was at −2.9 standard deviation score (SDS), at 7.6 yr height was at −3.99 SDS]. She showed hirsutism with sparing of her knees and elbows. The skin on her arms and legs showed increased tightening over the years, and similarly her face became affected. The lymphadenopathy spread and was present in the groins and axillae. Biopsies from both lymph nodes and subcutaneous tissue showed fibrofatty connective tissue with fibrosis and mononuclear cell infiltrates around the larger vessels. Inflammatory markers such as erythrocyte sedimentation rate (ESR) (160 mm/hr), and C-reactive protein (CRP) (62 mg/l) were constantly raised. Her liver and spleen were enlarged. At 7 yr, she developed increasing shortness of breath and lethargy. An echocardiogram showed mild left ventricular dilatation. A trial of oral prednisolone therapy caused some reduction in her ESR level (65 mm/hr), improved her clinical condition and activity levels and reduced lymphadenopathy. When steroid were weaned, her ESR again increased to 99 mm/hr. At the most recent physical examination at the age of 7.9 yr, her height was 101.7 cm (−4 SDS) and weight was 18.5 kg (−2.26 SDS). Her diarrhoea had settled on pancreatic enzyme replacement therapy. On prednisolone therapy, she was more energetic and less breathless. Her dermatosis had remained unchanged and she continued to have hepatomegaly (liver palpable 10 cm below the right costal margin) and splenomegaly (spleen palpable 2 cm below the left costal margin). Otherwise physical examination (including neuromuscular examination) gave normal results. She had a normal cognition. Ophthalmologic examination showed myopia with astigmatism but no signs of retinopathy. Audiometry showed a conductive hearing loss of 30 dB for which bilateral grommets were inserted. Her brain and spinal cord magnetic resonance imaging was normal, skeletal survey showed only mild sclerosis of the skull vault but otherwise gave normal results. Her karyotype was normal female, 46,XX. Immunological investigations revealed isolated immunoglobulin (Ig)A deficiency, raised IgG1, IgG2, and IgG3 with persistently raised ESR and CRP. Antibody screens for autoimmune disease were negative. She had an oral glucose tolerance test that showed elevated blood glucose with undetectable insulin concentration.

Gene and mutation identification Analysis of the single nucleotide polymorphism (SNP) microarray genotypes from six patients with PHID identified a single common 1.4 Mb region of shared homozygosity within cytogenetic band 10q22.1, between SNPs rs10509321 and rs1900515 (Fig. 1A). Inspection of the Ensembl and UCSC Genome Browsers demonstrated that the region included 14 known and predicted genes: ADAMTS14, C10orf27, CDH23, EIF4EBP2, LRRC2, KIAA1274, NODAL, NPFFR1, PCBD1, PPA1, PRF1, SLC29A3, SGPL1, UNC5B. Positional candidate genes were selected for sequencing in order to identify pathogenic mutations (Fig. 1B). Five different homozygous mutations were identified in the SLC29A3 gene encoding the equilibrative nucleoside transporter 3 (Fig. 1C, Table 1). Human ENT3 has been shown recently to be the cause of H syndrome, an autosomal recessive disorder with overlapping skin features to PHID (6), indicating that these are allelic disorders. hENT3 belongs to a structurally unique protein family of nucleoside and/or nucleobase transporters (13,14). The five homozygous mutations identified segregated with the disease within each family and were not present in over 100 Lebanese and Pakistani control chromosomes. Figure 1.Open in new tabDownload slideHomozygosity mapping and mutation results in five PHID families. (A) Homozygosity mapping of chromosome 10. Red bars indicate heterozygous single nucleotide polymorphisms (SNPs); blue bars, an extended run of homozygous SNPs greater than would be predicted by chance; green bars, a run of homozygous SNPs but insufficient to meet criteria for significance. (B) Genes within the 1.4 Mb interval bounded by heterozygous SNPs rs10509321 (CII.3) and rs1900515 (DII.1). (C) SLC29A3 sequence electrophoretograms demonstrating homozygous mutations in five PHID families. (D) Protein structure prediction of hENT3 by SVMTM software. (E) Multispecies alignment of the second transmembrane and the C-terminal cytoplasmic domains of ENT3 protein (accession numbers: Homo sapiens, NP_060814.4; Canis lupus, XP_546152.2; Bos taurus, XP_580640.3; Mus musculus, NP_076085.1; monodelphus domesticus, XP_001344438.1; Gallus gallus, XP_421594.2; Gasterosteus aculeatus, ENSGACP00000006741; Takifugu rubipens, ENSTRUP00000028258; Drosophila melangaster, FBpp0077753; Caenorhabditis elegans, NP_501807.3). Table 1.Mutation summary Mutation NM_018344.4 . Protein CCDS7310.1 . Protein domain . Predicted . Mutation predictions . Grantham . SIFT . c.347T>Gp.Met116ArgTransmembrane Domain 2Missense Hydrophobic to large basic910.03c.940delTp.Tyr314ThrfsX91Transmembrane Domain 7Frameshift––c.1309G>Ap.Gly437ArgIntra-cytoplasmic DomainMissense Small hydrophobic to large basic12512.0c.1330G>Tp.Glu444XIntra-cytoplasmic DomainNonsense––c.1346C>Gp.Thr449ArgIntra-cytoplasmic DomainMissense Small polar to large basic710.03Mutation NM_018344.4 . Protein CCDS7310.1 . Protein domain . Predicted . Mutation predictions . Grantham . SIFT . c.347T>Gp.Met116ArgTransmembrane Domain 2Missense Hydrophobic to large basic910.03c.940delTp.Tyr314ThrfsX91Transmembrane Domain 7Frameshift––c.1309G>Ap.Gly437ArgIntra-cytoplasmic DomainMissense Small hydrophobic to large basic12512.0c.1330G>Tp.Glu444XIntra-cytoplasmic DomainNonsense––c.1346C>Gp.Thr449ArgIntra-cytoplasmic DomainMissense Small polar to large basic710.03 Open in new tab Table 1.Mutation summary Mutation NM_018344.4 . Protein CCDS7310.1 . Protein domain . Predicted . Mutation predictions . Grantham . SIFT . c.347T>Gp.Met116ArgTransmembrane Domain 2Missense Hydrophobic to large basic910.03c.940delTp.Tyr314ThrfsX91Transmembrane Domain 7Frameshift––c.1309G>Ap.Gly437ArgIntra-cytoplasmic DomainMissense Small hydrophobic to large basic12512.0c.1330G>Tp.Glu444XIntra-cytoplasmic DomainNonsense––c.1346C>Gp.Thr449ArgIntra-cytoplasmic DomainMissense Small polar to large basic710.03Mutation NM_018344.4 . Protein CCDS7310.1 . Protein domain . Predicted . Mutation predictions . Grantham . SIFT . c.347T>Gp.Met116ArgTransmembrane Domain 2Missense Hydrophobic to large basic910.03c.940delTp.Tyr314ThrfsX91Transmembrane Domain 7Frameshift––c.1309G>Ap.Gly437ArgIntra-cytoplasmic DomainMissense Small hydrophobic to large basic12512.0c.1330G>Tp.Glu444XIntra-cytoplasmic DomainNonsense––c.1346C>Gp.Thr449ArgIntra-cytoplasmic DomainMissense Small polar to large basic710.03 Open in new tab Two novel homozygous mutations predicted to cause premature protein truncation were identified, a 1 bp deletion c.940delT and a nonsense mutation c.1330G>T. The deletion causes a frame shift mutation p.Y314Tfs in transmembrane domain 7 and is predicted to result in the substitution of transmembrane domains 8–11 with an irrelevant protein sequence of 91 amino acids derived from an alternate reading frame. The nonsense mutation p.E444X is predicted to lead to premature protein truncation with loss of part of the final cytoplasmic domain and transmembrane domain 11 of hENT3. It would appear unlikely that the either residual protein would be capable of nucleoside transport function.Three homozygous missense mutations of SLC29A3 were identified to involve conserved amino acids (Table 1). The first missense mutation involves Methionine116 in transmembrane domain 2, which is a hydrophobic amino acid in species as distantly related as humans and nematodes, with a large basic amino acid, Arginine (p.M116R; Fig. 1C and E). The Grantham score for this substitution is 91, which is classified as a moderate change (15). Analysis using the SIFT prediction software also classifies this as a likely deleterious change (16). The introduction of a large basic amino acid into a transmembrane domain would likely result in significant disruption of the hydrophobic nature of the domain and result in retention of the transporter protein within the endoplasmic reticulum, as has been demonstrated for other membrane proteins (17).The second missense change is a recently reported Glycine to Arginine substitution at hENT3 amino acid 437 (p.G437R Fig. 1C), which is located in the putative cytoplasmic domain between transmembrane domains 10 and 11. This is the only mutation in the PHID cohort that is shared with the known mutations in H syndrome. Of particular interest is the observation that this is the sole individual in the PHID cohort who has not developed insulin-dependent DM. The p.G437R change involves the substitution of a large basic amino acid at a position where 41 members of the ENT family as distantly related as humans and Arabidopsis thaliana encode a small hydrophobic amino acid (Fig. 1E) (18). The Grantham difference for this change is 125 and is classified as a moderately radical change.The third missense change occurs at hENT3 amino acid residue 449, which is also located in the final putative intracellular domain between transmembrane domains 10 and 11, and involves the substitution of a Threonine by an Arginine (p.T449R). The amino acids encoded at this position in hENT3 orthologs are small polar/uncharged or small hydrophobic residues (Fig. 1E). Substitution by arginine is associated with a Grantham score of 71 and is predicted by SIFT as not to be tolerated.