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Exome sequencing in the diagnosis of an atypical phenotype of infantile hyalinosis

Temtamy, Samia A.; Aglan, Mona S.; Otaify, Ghada A.; Abdel-Hamid, Mohamed; Ismail, Somaia; Makrythanasis, Periklis; Hamamy, Hanan; Antonarakis, Stylianos E.

Middle East Journal of Medical Genetics: January 2015 - Volume 4 - Issue 1 - p 18–23
doi: 10.1097/01.MXE.0000457060.97665.02
Original articles

Introduction Farber lipogranulomatosis and infantile systemic hyalinosis are two rare autosomal recessive diseases with overlapping phenotypic features.

Aim The aim of the study was to present the difficulty in differentiating between these two rare disorders on clinical bases only and emphasize the importance of exome sequencing in the accurate diagnosis of patients with atypical presentations of known genetic diseases.

Patients and methods Two Egyptian cousins born to consanguineous parents presented with hoarseness of cry, painful swollen joint contractures, failure to thrive, diffuse thickening and hyperpigmentation of skin over bony prominences, and death before 2 years of age. Their initial differential diagnosis of Farber disease was ruled out because no pathogenic mutations were identified in the ASAH1 gene in either the proband or the parents. Accurate diagnosis in the affected was revealed as infantile systemic hyalinosis by exome sequencing, showing a homozygous pathogenic variant in the ANTXR2 gene.

Conclusion In the present work we highlight the need for molecular studies by next-generation sequencing for accurate diagnosis, prevention, and proper counseling when genetic diagnosis cannot be reached by other conventional methods.

Departments of aClinical Genetics

bMolecular Medical Genetics, Division of Human Genetics and Genome Research, Center of Scientific Excellence of Human Genetics, National Research Centre, Cairo, Egypt

cDepartment of Genetic Medicine and Development, University of Geneva

dService of Genetic Medicine, University Hospitals of Geneva

eiGE3, Institute of Genetics and Genomics of Geneva, Geneva, Switzerland

* Samia A. Temtamy and Periklis Makrythanasis contributed equally to the writing of this article.

Correspondence to Ghada A. Otaify, MD, PhD, Department of Clinical Genetics, Division of Human Genetics and Genome Research, Center of Scientific Excellence of Human Genetics, National Research Centre, El-Buhouth St, Dokki, 12311 Cairo, Egypt Tel: +201001788870; fax: +20 233 370 931; e-mail:

Received October 10, 2014

Accepted October 23, 2014

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Juvenile hyaline fibromatosis (JHF) (OMIM 228600) and infantile systemic hyalinosis (ISH) (OMIM 236490) are autosomal recessive disorders of the connective tissue that are characterized by abnormal growth of hyalinized fibrous tissue that involves the skin and other organs (gingiva, joints, and bones) leading to multiple subcutaneous skin nodules and/or pearly papules, gingival hypertrophy, flexion contractures of the joints, and osteolytic bone defects (El-Maaytah et al., 2010; Slimani et al., 2011; Denadai et al., 2012). The onset of clinical manifestations of JHF is normally in the first 3–4 months of life, and ISH typically manifests at an earlier age in the first weeks to months of life with visceral involvement and early lethality (Félix et al., 2004; Mendonça et al., 2011). In both disorders, mental development is normal (Jaouad et al., 2014). In JHF, lesions may be nodular and/or papular skin lesions (on the face, neck, and especially on retroauricular and perinasal regions), large tumors (especially on the scalp, trunk and limbs), or perianal plaques or nodules. Gingival hypertrophy is common and sometimes impairs eating. Other manifestations include osteolytic lesions (especially in the distal phalanges and metaphyses), cortical thinning, and generalized osteopenia.

In ISH, in addition to cutaneous lesions, there are articular contractures, gingival hypertrophy, and bone abnormalities, and the skin is thick, with hyperpigmentation over bone prominences. The disease progresses with persistent diarrhea, recurrent infections, and death within the first 2 years of life (Lindvall et al., 2008; Jaouad et al., 2014). Systemic involvement has been reported in severe cases and is usually associated with a fatal outcome. Infiltration of the small intestine and colon, the most common form of systemic involvement, leads to malabsorption and protein-losing enteropathy with diarrhea, failure to thrive, and an increased susceptibility to infection (Lindvall et al., 2008; Al-Mubarak et al., 2009). Other organs that may be affected include the heart, trachea, esophagus, stomach, spleen, adrenal glands, thyroid, lymph nodes, and skeletal muscle (Al-Mubarak et al., 2009).

Histopathological findings in JHF and ISH are characteristic and identical. Deposits of abundant homogenous eosinophilic material are observed in the papillary and reticular dermis. Embedded in this ground substance are numerous spindle-shaped fibroblasts (Urbina et al., 2004). Electron microscopy shows similar features in both conditions in the form of numerous vesicles or vacuoles filled with fibrogranular-banded material that is sometimes deposited around vessels and in the extracellular matrix (Stucki et al., 2001; Larralde et al., 2001). In ISH, hyaline deposition is more widespread than in JHF and can affect many tissues including skin, skeletal muscle, cardiac muscle, gastrointestinal tract, lymph nodes, spleen, thyroid, and adrenal glands (Nofal et al., 2009).

JHF and ISH are two variants of the same disease, now jointly called hyaline fibromatosis syndrome (HFS) (Hanks et al., 2003; Nofal et al., 2009; El-Kamah et al., 2010; Denadai et al., 2012). The disease was first called molluscum fibrosum, and then JHF and ISH (Drescher et al., 1967). Since the realization that these are different manifestations of the same disease, the unifying term HFS has been adopted (Nofal et al., 2009). Six Egyptian patients with HFS were reported by El-Kamah et al. (2010) in two unrelated families.

Denadai et al. (2012) proposed a four-grade scoring system of the disorder to reflect increasing severity, with grade 4 resulting in early death due to severe clinical decompensation.

Different mutations (missense, frameshift, in-frame, nonsense, and splice site) of the anthrax toxin receptor-2 (ANTXR2) (OMIM 608041) gene, also known as the capillary morphogenesis protein gene-2 (CMG2) and located on chromosome 4q21, have been found to be responsible for both disorders (Dowling et al., 2003; Hanks et al., 2003; Denadai et al., 2012). ANTXR2 encodes a transmembrane protein in which the von Willebrand A domain binds to both lamin and collagen IV, suggesting that this protein plays a role in basement membrane matrix assembly and endothelial cell morphogenesis (Hanks et al., 2003; El-Maaytah et al., 2010). Hanks et al. (2003) suggested that a defect in ANTXR2 can lead to extravasation of hyaline material (plasma components) through the basement membrane into the perivascular space. This phenomenon may explain the histological findings of both JHF and ISH (Hanks et al., 2003; El-Maaytah et al., 2010).

Farber disease, also known as Farber’s lipogranulomatosis (OMIM 228000), is a clinically heterogenous autosomal recessive disease caused by deficiency of lysosomal acid ceramidase due to mutations in the ASAH1 gene that encodes the acid ceramidase enzyme leading to a unique triad of subcutaneous nodules, painful and progressively deformed joints, and hoarseness due to laryngeal involvement (Alves et al., 2013). On the basis of age at onset, the severity of symptoms, and the difference in organ affection by ceramide storage, seven disease subtypes have been discerned (Levade et al., 2009).

The present report describes two Egyptian cousins with an atypical clinical presentation whose diagnosis was revealed as ISH by next-generation sequencing.

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Clinical report

An Egyptian consanguineous family with three affected patients (two boys and one girl) was referred to the Limb Malformations and Skeletal Dysplasia Clinic, National Research Centre, due to repeated birth of infants with painful stiff flexed fingers, knee and elbow joints and skin discoloration over bony prominences. In addition to the proband, there were two other similarly affected family members, including a half female sibling who died at 10 months of age and a male cousin who was seen and examined by our team before his death at 9 months of age. The family pedigree is presented in Fig. 1.

Figure 1

Figure 1

Patient 1 (proband): this was a 7-month-old boy, born at full term by cesarean section to an apparently healthy consanguineous couple, a 22-year-old mother and a 35-year-old father, after two spontaneous abortions at 2 and 3 months’ gestation, respectively. His birth weight was 2 kg and he had a history of oligohydramnios. The main complaints were painful joint stiffness with dark discoloration of skin over bony prominences. On examination, the patient was seen to have limited extension of elbows, interphalangeal joints, knees, and ankles with painful movements of all joints. Diffusely thickened skin with small nodules was noted over the proximal phalanges of toes and hyperpigmentation opposite all knuckles and at the medial side of ankles (Fig. 2a–c). Orodental examination revealed macrostomia, partial ankyloglossia, and prominent philtrum. His cognitive development was normal. His height was 57.5 cm (−3.6 SD), weight was 6.5 kg (−1.8 SD), and head circumference was 40 cm (−2.0 SD). The patient died at 10 months of age because of persistent vomiting, diarrhea, chest infection, and dehydration.

Figure 2

Figure 2

There was a history of a similarly affected half paternal female sib who died at 10 months of age, and a similarly affected male cousin (patient 2) who died at 9 months of age from vomiting, diarrhea, dyspnea, and septicemia. This male cousin was seen by our clinical team. He also had painful joint contractures, with knee joint swelling, mild gingival hypertrophy, hyperpigmentation over the forehead and joints, and thick skin nodules at interphalangeal joints (Fig. 2 d–f). He developed renal stones at 5 months of age and his urine analysis revealed urate crystals; however, his kidney functions were normal. The patient died before blood samples could be taken for molecular studies, but samples were taken from his parents. Neither of our patients had fibrous nodules or papillomas of the lips, scalp, or in postauricular areas.

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Informed consent was obtained from the parents of affected cases before implementation of the molecular studies. Peripheral blood was collected from the affected child and his parents and from parents of his deceased cousin.

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Extraction of genomic DNA and exclusion of ASAH1 gene mutations

Genomic DNA was extracted from peripheral blood lymphocytes of patient 1, his parents, and the parents of patient 2 after receiving signed informed consent according the guidelines of the Medical Research Ethical Committee of the National Research Centre. The entire coding region of the ASAH1 gene was amplified using specific primers designed using Primer3 Input Software (version 0.4.0). The sequence of primers is available upon request. PCR products were purified by the QIAquick PCR Purification Kit (Qiagen, Germany) and directly sequenced in both directions using the Big Dye Termination Kit (Applied Biosystems, Foster City, California, USA) and analyzed on the ABI Prism 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instructions.

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Exome sequencing

DNA from the patient was used for exome sequencing as described previously (Makrythanasis et al., 2014a, 2014b). Briefly, exome was captured using the SureSelect Human All Exons v5 reagents (Agilent Inc., Santa Clara, California, USA). Sequencing was performed in an Illumina HiSeq 2000. The exome library was indexed, separated into two equal halves, and sequenced in two different lanes. Four half-libraries were sequenced in each HiSeq lane. The raw results were analyzed using a custom pipeline that utilizes published algorithms in a sequential manner (BWA for mapping the reads, SAMtools for detection of variants, Pindel for the detection of indels, ANNOVAR for the annotation). The entire coding sequence corresponding to the RefSeq coding genes was used as the reference for the calculation of coverage and reads on target. All experiments were performed using the manufacturer’s recommended protocols without modifications. The deletion was confirmed through Sanger sequencing in the affected. As expected, the parents were heterozygous for the duplicated nucleotide and the frame shift was readily seen at the Sanger sequencing (Fig. 3).

Figure 3

Figure 3

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No pathogenic mutations were identified in the ASAH1 gene in the proband or in the parents, ruling out Farber disease. Exome sequencing identified a 1-bp duplication in the ANTXR2 gene (NM 058172.5). The likely pathogenic variant was c.1190dupG (p.Asp398*). Which creates a frameshift and a stop codon, posing the diagnosis of infantile hyalinosis.

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Our patients presented atypical manifestations of ISH, as they revealed hoarseness of cry, painful swollen joint contractures, bony prominences over interphalanges and other joints, failure to thrive, short stature, diffuse thickening of skin, which was hyperpigmented over bony prominences, and death before 2 years of age (similar to Farber disease). They did not have any papules on the face or lips, nor severe gingival hyperplasia, and no fibromas in the scalp or fleshy nodules in the perianal or postauricular areas, which are characteristic features of ISH (El-Kamah and Mostafa, 2009; Nofal et al., 2009).

Infantile Farber disease classically presents with a triad of progressive painful deformed joints, subcutaneous nodules, and hoarseness of voice. Joints are generally the first to be affected. Subcutaneous nodules present a hallmark of the disease and should be present in all types except neonatal-visceral cases and in patients with prosaposin deficiency (Levade et al., 2009).

The main phenotypic differences and similarities between Farber lipogranulomatosis and infantile hyalinosis as compared with our patients are shown in Table 1. As symptoms appeared very early in the neonatal period we assume that other manifestations could have appeared if our patients had survived longer.

Table 1

Table 1

Our patients had many clinically overlapping features with subtype 4 of Farber lipogranulomatosis, similar to the two sibs previously reported by Antonarakis et al. (1984). Phenotypic overlap between Farber disease and variants of infantile hyalinoses was also noted by Moser et al. (1989).

Because of the marked phenotypic overlap between our patients and cases of Farber lipogranulomatosis, the DNA of our patients was analyzed to rule out any mutations for the ASAH1 gene, which was found negative. Subsequently, the patients’ DNA was subjected to whole-exome sequencing, which revealed the presence of the ANTXR2 gene mutation, thus confirming a diagnosis of ISH.

More than 150 cases of HFS have been reported and 34 different mutations, from exon 1 to exon 15, have been identified (El-Kamah et al., 2010; Deuquet et al., 2011; Denadai et al., 2012; Jaouad et al., 2014). Among them, exon 13 is a hotspot for frameshift mutations, which include insertion of one or two bases (c.1073–1074insC and c.1073–1074insCC) and deletion of one base (c.1074delT). Yan et al. (2013) have shown that these three frameshift mutations represent ∼60% of all pathogenic alleles. The frequency of insertions and deletions at positions 1073–1074 is likely due to its proximity to a low complexity, GC-rich, region encoding a stretch of proline codons that could constitute a vulnerable site for errors, during DNA replication. The c.1047delT mutation was described in four unrelated families of various ethnic groups with both ISH and JHF (Hatamochi et al., 2007; Huang et al., 2007; El-Kamah et al., 2010). Of note, El-Kamah et al. (2010) reported a large Egyptian pedigree with three affected children with JHF carrying this recurrent mutation. The novel c.1190dupG mutation identified in our family is located in the cytoplasmic domain of the predicted protein and causes a frameshift and a premature termination codon 1 amino acid downstream (p.Asp398*). Nonesense/frameshift mutations are assumed to disrupt protein function through synthesis of truncating proteins.

Our results support the notion that whole-exome sequencing is becoming the method of choice to identify pathogenic gene mutations in patients with atypical manifestations for known genetic disorders.

The precise diagnosis of the disease is an essential step for proper family planning and future pregnancies as prenatal detection can now be offered to couples at risk. In addition, the carrier status of additional family members can be provided.

Little is known about the consequences of mutations in the ANTXR2 gene, and not only the site of a mutation but also the nature of the insertion and/or deletion is important – for example, the change in the reading frame. Therefore, it is important to perform genotype–phenotype correlation at the mRNA and protein levels and analyze the molecular consequences of the specific mutations of the ANTRX2 gene to evaluate potential therapeutic targets (Jaouad et al., 2014).

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Conflicts of interest

There are no conflicts of interest.

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anthrax toxin receptor-2 protein; ANTXR2; cell morphogenetic protein G-2; CMG2; consanguinity; genetic counseling; infantile systemic hyalinosis; next-generation sequencing

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