A 37-kb Deletion in Region 16p13.3 in an Infant with Osteopetrosis and Congenital Diarrhea Including the CLCN7 and PERCC1 Genes Open Access

Congenital diarrhea and enteropathies (CODEs) are rare genetic disorders characterized by persistent diarrhea, lasting more than 2 weeks, and presenting in the first weeks of life. CODEs are often associated with feeding intolerance, malabsorption, and congenital anatomic anomalies. CODEs are less common in infants than other diarrheas, which are mostly due to allergic or infectious agents. It is difficult to define the etiology of CODEs in this group, but with the uptake of next-generation sequencing and genome-wide studies, the rate of genetic diagnosis of CODEs is increasing [1].

The PERCC1 gene, also known as proline- and glutamate-rich protein with coiled-coil domain 1, is a recently annotated gene that plays a role in the development of enteroendocrine cells and the secretion of enteroendocrine hormones. PERCC1 was recently found to be related to autosomal recessive congenital diarrhea 11 (OMIM #618662). Oz-Levi et al. [2] showed that this gene flanks a noncoding sequence termed the intestine-critical region (ICR), which controls gastrointestinal expression of PERCC1. The authors also demonstrated that the absence of the ICR, resulting in gastrointestinal expression of PERCC1, caused intractable malabsorptive diarrhea in infancy.

Osteopetrosis (OP) is a genetic disease that occurs as a result of disruption to osteoclast activity. It is characterized by bone fractures; optic nerve compression and associated visual loss due to skull base sclerosis; thrombocytopenia and anemia due to the absence of bone marrow cavity; hypocalcemia; and osteomyelitis. OP can be inherited as an autosomal recessive, dominant, or X-linked trait, with the most severe forms being autosomal recessive [3]. Autosomal recessive OP (AROP) has a variable presentation, most commonly including failure to thrive, hypocalcemia, seizures, hepatosplenomegaly, hydrocephalus, vision or hearing loss, and cytopenias [4]. There are eight AROP, related to the CA2, CLCN7, OSTM1, PLEKHM1, SNX10, TCIRG1, TNFSF11, and TNFRSF11A genes, and three autosomal dominant OP (ADOP), related to the CLCN7, LRP5, and PLEKHM1 genes. As can be noted here, the CLCN7 gene is associated with both AROP and ADOP. OP, autosomal recessive 4 (AROP4), is caused by homozygous or compound heterozygous pathogenic variants in the CLCN7 gene, where both alleles carry variants leading to a complete loss of chloride channel function (OMIM #611490). In contrast, OP, autosomal dominant 2 (ADOP2), is often associated with monoallelic dominant-negative variants in the CLCN7 gene, which produce a dysfunctional protein, resulting in impaired chloride channel function and bone resorption (OMIM #166600) [5, 6]. Herein, we report a patient presenting OP and congenital diarrhea, with a homozygous deletion in the 16p13.3 region containing the CLCN7 and PERCC1 genes.

This case concerns a female born at 37 weeks’ gestation, with a birth weight of 3.240 kg (75th–90th centile), who was delivered in the hospital by caesarian section to a 19-year-old mother. Her birth length was 52 cm (90th centile), and OFC was 34 cm (50th–75th centile). She is the first child of healthy, consanguineous parents. Prenatal screening tests for infection were unremarkable. The infant’s Apgar scores were 8 and 10 at delivery. She presented on day 1 of illness with a purpuric rash and frequent, watery stools. Examination revealed fever (38.1°C), tachycardia, dehydration, and jaundice, but the patient was otherwise normal. Her platelet count was 33,000/mm³ in the complete blood count, and her C-reactive protein and erythrocyte sedimentation rate were normal. At a follow-up, she had non-formed, blood- and mucus-free defecation (formed as Bristol 7) [7] 10–12 times a day. Stool tests, including the standard culture assessment of enteric bacterial pathogens and the tests of viral pathogens such as rotavirus, cytomegalovirus, norovirus, and adenovirus using PCR and/or ELISA, were negative. No erythrocytes or leukocytes were found in stool microscopy, the stool pH was 6, and it was negative for fat and the reducing substances.

An evaluation for metabolic diseases and standard serum testing, including the complete blood count (except for thrombocytopenia), electrolytes, erythrocyte sedimentation rate or C-reactive protein, liver function tests, lipid panel, immunoglobulin G and A (IgG and IgA), and T- and B-cell subsets, were normal at the follow-up. Noninfective, nonsecretory, mixed-type chronic osmotic diarrhea was considered after observing a decrease in diarrheal volume during a trial of withholding feeds for a minimum of 24 h. There were no significant improvements during trials of feeds with a carbohydrate-free formula, extensive hydrolyzed formula, or amino acid-based formula. Total parenteral nutrition was given because of weight gain insufficiency and diarrhea. Endoscopy could not be performed.

Meanwhile, the patient had an increased bone density on an X-ray (shown in Fig. 1), and ophthalmoscopy revealed pale optic discs in both eyes, consistent with OP. However, since the diagnosis of OP did not explain the diarrhea, clinical exome sequencing was carried out, with a suspicion of CODE in the patient.

Clinical exome sequencing was performed using the SOPHiA™ GENETICS CESv_2 kit on the Illumina NextSeq 500 platform. The raw data were analyzed and annotated, and filtering steps were performed using SOPHiA™ DDM (SOPHiA Genetics^®). Copy number variation analyses, also conducted using SOPHiA™ DDM, revealed a homozygous exon 2–25 deletion in the CLCN7 gene related to OP. No variant associated with congenital diarrhea was detected in single nucleotide variant analysis. To confirm the homozygous deletion of the CLCN7 gene, a chromosomal microarray was performed using Agilent GenetiSure Cyto CGH Microarray 8 × 60K Kit (Santa Clara, CA, USA). This method enabled us to detect a homozygous 39-kb deletion on chromosome 16p13.3 ([hg19] (1,476,688-1,516,085)x0) which included three genes in addition to the CLCN7 gene: C16orf91, CCDC154, and PERCC1 (shown in Fig. 2). The homozygous deletion of the CLCN7 gene was found to be compatible with AROP, and the deletion of the PERCC1 gene explained the patient’s presentation with chronic diarrhea. Microarray analysis performed on the parents showed that they carried this deletion as heterozygous.

CODEs are generally monogenic, and they can be divided into genetic variants that directly affect the intestinal epithelium or the immune system. However, in some CODE cases, genetic causes cannot yet be determined [1]. The diagnostic rates are 71.9% (46/64) for targeted gene panel sequencing and 57.5% (42/73) for exome sequencing (p = 0.081) [8]. In a study from 2019, Oz-Levi et al. showed that five out of eight patients, from seven unrelated Iraqi Jewish families with the autosomal recessive severe congenital malabsorptive diarrhea pattern, had a deletion in a sequence, including a noncoding sequence called the ICR, which controlled gastrointestinal expression of PERCC1 gene on chromosome 16 between the C16orf91 and CCDC154 genes.

The present patient had congenital diarrhea with OP, and molecular analysis revealed a deletion in the 16p13.3 region containing the OP-related CLCN7 gene and the PERCC1 gene, which has recently been associated with congenital diarrhea. Deletion of the 16p13.3 region including the CLCN7 and PERCC1 genes is extremely rare. Previously, there was only 1 case with a microdeletion including the CLCN7 gene, which was presented by Pangrazio et al. in 2012 [9]. The patient had a similar contiguous gene deletion in the chromosome 16p13.3 region, including 4–11 exons of GNTPG, UNKL, C16orf91, and CCDC154 and exons 7–25 of the CLCN7 gene. He had classical AROP findings such as infantile presentation of increased bone density, failure to thrive, and pancytopenia, and he also had chronic diarrhea. The diarrhea was linked to pseudomembranous colitis, which suggested a partial and secondary immune deficit. This patient passed away due to sepsis at the age of 6 months, and no autopsy was performed. In the literature, no isolated large deletion of the CLCN7 gene has yet been reported. However, truncating variants expected to cause loss of function in this gene have been frequently identified, and diarrhea has not been reported in these patients [10]. As mentioned above, in the study of Oz-Levi et al. (2019), it was revealed that the finding of congenital diarrhea in this patient, as in our case, was related to the PERCC1 gene in the deletion region.

Therefore, the present instance represents the second patient diagnosed with OP and congenital diarrhea, accompanied by a deletion in the 16p13.3 region encompassing the CLCN7 and PERCC1 genes. In conclusion, the presented case confirms the recently identified critical region on chromosome 16 associated with congenital diarrhea, and it demonstrates that 16p13.3 deletions should be considered in patients with congenital diarrhea and OP, as well as in patients with isolated congenital diarrhea.

Written informed consent was obtained from the parent/legal guardian of the patient for publication of the details of their medical case and any accompanying images. Ethical approval was not required for this study in accordance with local/national guidelines.

The authors have no conflicts of interest to declare.

There was no funding relevant to this study.

S.S. was involved in the conception of the work and collation of clinical data and contributed to writing the original draft. G.K. contributed to the conception, data analysis, interpretation, and critically revised the manuscript. D.K. was involved in collating clinical data and provided critical review and editing of the manuscript. E.V., A.K., A.K., M.T.E., T.D., S.D., and S.H. contributed to the collation of clinical data. Y.B. contributed to the conception and data analysis and was involved in writing both the original draft and in the review and editing process.

Semih Sandal and Gulsum Kayhan contributed equally and share the first authorship.

The raw data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to patient privacy.