Exome sequencing identifies a novel TTC37 mutation in the first reported case of Trichohepatoenteric syndrome (THE-S) in South Africa

The index patient was a male of Somalian descent, born to consanguineous parents (first cousins once-removed, i.e. fourth degree relatives) living in South Africa (see Fig. 1 for pedigree). He was born at 35 weeks gestation, weighing 1940 g (3rd percentile) and remained in hospital initially for three weeks for neonatal jaundice and poor weight gain. The parents noted subtle patchy hyperpigmentation after he completed phototherapy for jaundice. His hair was shaved per their custom and was reportedly not unusual. On discharge he had received all routine vaccinations including live BCG and oral Polio drops.

At age three months the patient presented with an acute history of respiratory distress. In addition, he had failed to thrive and his mass was 2600 g (<<3rd centile for corrected age). There was clinical evidence of a pneumonia and the liver edge was palpable 2 cm below the costal margin. Patchy, poorly circumscribed areas of hyperpigmentation were present on the trunk and limbs (Fig. 2a). There was an exfoliating rash particularly on the hands and feet with erythema of the palms and soles suggestive of erythroderma (Fig. 2b). The forehead was prominent (Fig. 2c), giving a triangular appearance to the face, but no hypertelorism or other dysmorphic features were noted.

An echocardiogram was performed and found to be normal, while chest x-ray showed an extensive bilateral interstitial pneumonitis with alveolar changes (Fig. 2d). The C-reactive protein (CRP) increased over 3 days from 9 to 43 mg/L (ref: 0–10). Tracheal aspirate was positive for cytomegalovirus (CMV) but not other respiratory viruses, and CMV viraemia was documented with a viral load of log 5.97. Blood, sputum and stool culture showed no evidence of bacterial infection. The full blood count was normal, with normal number of lymphocytes. On subset analysis, the natural killer (NK) cell count was 81/microliter (ref: 300–700) and the NK cell percentage was 3% (ref: 8–17) (Table 1). There was also a low serum IgG levels of <1.41 g/L (ref 3.00-10.00) but normal IgA and IgM. Liver functions showed very elevated gamma-glutamyl transferase (942 IU/L, ref: 2–30); somewhat elevated conjugated bilirubin (10 micromol/L, ref: 0–6), alanine transaminase (39 IU/L, ref: 4–35) and aspartate transaminase (75 IU/L, ref: 0–65); low albumin (23 g/L, ref: 28–46), and normal alkaline phosphatase (180 IU/L, ref: 75–316). The plasma amino acid profile was normal, as were urinary organic acids. Skin biopsy histology of a hyperpigmented macule showed abundant melanin but no specific features for pathological processes such as graft versus host disease.

The patient was treated in the intensive care unit. Treatment included ventilation with a high frequency oscillator, intravenous ganciclovir, and other medications as appropriate. During the admission diarrhoea was noted, but was not a prominent feature. Following three weeks of intensive therapy, the patient died of ongoing severe pneumonitis. Supervening nosocomial sepsis was suspected but unproven. The family declined an autopsy. The clinical suspicion was of a combined immune deficit, with a family history that suggested an autosomal recessive or perhaps X-linked disorder.

A male sibling born 4 years earlier, weighed 1840 g (<<3rd centile) at term gestation. He was treated for neonatal jaundice and subsequently readmitted at two months of age with chronic diarrhoea, dehydration and failure to thrive. He was noticed to have hair that was “sticking out”, and both hyper- and hypopigmented skin macules. Other clinical features included a prominent forehead with a triangular face and low-set ears, penile hypospadias and bilateral camptodactyly of the fingers. Further laboratory investigations confirmed a secretory diarrhoea with albumin loss and metabolic acidosis. Endoscopic biopsies of the stomach and duodenum were performed. The stomach histology was normal, whereas the duodenal biopsy showed flattening and thickening of some villi, and preserved crypt architecture; in addition the lamina propria contained a sprinkling of lymphocytes and plasma cells. The liver function test was only minimally deranged. The full blood count, lymphocyte subsets and serum immunoglobulin profile were similar to that of the index case, except that the IgA was also low. He remained in hospital until his demise at 5 months of age, following documented episodes of Klebsiella and Enterococcal septicaemia. Additional investigations showed a normal karyotype, and normal profile of urine organic acids and urine and blood amino acids. No definitive diagnosis was made, and some details only became available to us after the death of the index case. Phenotypic comparisons between the index case and his deceased brother are shown in Table 1. The couple also has a healthy daughter, and a female stillbirth for reasons which were considered to be unrelated (Fig. 1).

Prior to demise of the index case, the parents were counselled on a possible genetic immunodeficiency and consented for blood samples from all live members of the family to be submitted for exome sequencing.

The study was approved by the Health Research Ethics Committee of Stellenbosch University (study no. N13/05/075). Parents gave informed consent to participate in the study, which included the genetic evaluation of their children. Additional informed consent was obtained from the parents to include photographs in this article. The study adhered to the ethical guidelines as set out in the “Declaration of Helsinki, 2013” [20]. Venous blood for DNA extraction and WES was drawn from the index case (1 ml) and both of his parents (5 ml). Blood was also drawn from the unaffected sister for genetic analysis. DNA was purified from blood using the Nucleon BACC3 Kit (Amersham Biosciences, Buckinghamshire, UK). DNA from a formalin-fixed duodenal biopsy sample of the previously deceased male sibling was extracted using the QIAamp® DNA FFPE Tissue Kit (Qiagen, Hilden, Germany).

Library preparation for sequencing was carried out using the Ion AmpliSeq™ Exome RDY Kit and the Ion Xpress™ Barcode Adaptors 1–16 Kit (Life Technologies, Carlsbad, California, United States). The DNA template for sequencing was prepared on the Ion Chef system using the Ion PI™ Hi-Q™ Chef Kit and the Ion PI™ Chip Kit v3. Sequencing was carried out on the Ion Proton™ (Thermo Fisher, Carlsbad, California, United States) at the Centre for Proteomic and Genomic Research (CPGR), Observatory, Cape Town, South Africa.

Sequences were aligned to the human reference genome, hg19 using Torrent Mapping Alignment Program (TMAP, version 4.4.11-1) in the ion-analysis workflow on the Torrent Suite (version 4.4.3). Base quality score recalibration, indel realignment and variant calling were performed using the variantCaller (version 4.4.3.3) plugin on the Torrent Suite and variant annotation was performed. Variant prioritization was performed using TAPER^TM, a custom-designed, in-house method and variants in a homozygous recessive state or compound heterozygous state were prioritized as potential candidate variants [21]. In brief, TAPER™ 1) submits VCF files to an online variant caller, 2) removes all synonymous and non-frameshift indel variants, 3) removes all variants with a frequency of greater than 1% if present in the 1000 Genomes Project or Exome Sequencing Project 6500, 4) removes all variants with negative Genomic Evolutionary Rate Profiling (GERP) +++ scores, 5) removes all variants with Functional Analysis through Hidden Markov Models (FATHMM) scores greater than 0.1, 6) removes X and Y chromosome variants (if the pedigree does not indicate sex-linked inheritance) and lastly 7) identifies associated disorders for prioritised genes. Additionally, to validate findings obtained using TAPER™, variant filtering and prioritization was also done using Ion Reporter™ Software (version 5.0).

Using custom scripts, the VCF files for both the mother and father were merged to create a multi-sample VCF file. This file was subsequently filtered for single nucleotide substitutions that were flagged “PASS” by a custom variant caller filter. Identity by descent (IBD) was calculated uing PLINK (http://​zzz.​bwh.​harvard.​edu/​plink/​) [22]. IBD was estimated in order to determine the degree of relatedness across the two individuals (PI(HAT)). This is calculated by using the inferred probabilities of individuals sharing an allele. Individuals may therefore share no alleles, one allele or both alleles. Following IBD, the runs of homozygosity were calculated to identify which segments of specific chromosomes are identical to one another. This allowed further inference of IBD as these segments were identical as a result of inheritance as opposed to sequence similarity.

A 239 base pair fragment containing the candidate variant was polymerase chain reaction (PCR)-amplified from genomic DNA of the index case, his affected brother, his unaffected parents and his unaffected sister using the following primers: TTC37F- 5′ AATCATAATCAGACACTACATCTGC-3′ and TTC37R- 5′ GCTCATAGTCATCTTTGGCATATAA. Each amplicon was bi-directionally sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Perkin-Elmer, Applied Biosystems Inc., Foster City, California, USA.), followed by electrophoresis on an ABI 3130XL Genetic Analyzer (Perkin-Elmer, Applied Biosystems Inc., Foster City, California, USA). All automated DNA sequencing reactions were performed at the Central Analytical Facility at Stellenbosch University, Stellenbosch, RSA.