A Novel Frameshift Homozygous Mutation in DHCR7 with a Known Missense Homozygous Mutation in the PROC in a 6-Year-Old Boy: A Child with Two Rare Genetic Diseases

Abstract

In the present case report, we described a 6-year-old-boy with developmental delay, mental retardation, lack of speech, skin scars, and 2 to 3 toe syndactyly from healthy consanguineous Turkish parents. The whole exome sequencing (WES) analysis of this patient showed homozygous variant c.418T > C p.(Cys140Arg) in PROC gene and novel homozygous variant c.57dupC p.(Asn20Glnfs*2) in the DHCR7 gene. This finding demonstrated that WES is of great value for the diagnosis of two separate genetic disorders in a patient with multiple dysmorphic and other clinical features. It should also be kept in mind that the coexistence of two autosomal recessive diseases could be observed in highly related consanguineous marriages. The combined evaluation of clinical and laboratory data provided extremely valuable insight into the diagnosis of this unique case.

Introduction

Smith–Lemli–Opitz syndrome (SLOS; 270400) is an autosomal recessive disease characterized by mental retardation, developmental delay, ptosis, structural heart defects, urogenital problems, behavioral manifestations, central nervous system abnormalities, and the hallmark sign of 2 to 3 toe syndactyly. SLOS is caused by a homozygous mutation of the DHCR7 gene on chromosome 11q13.4. ^{1 2} Protein C (PC; 612304) is coded by the PROC gene on chromosome 2q14.3 and comprises nine exons. PC is prompted by the thrombin–thrombomodulin complex and applies anticoagulant activity. Drastic congenital PC deficiency is associated with manifest thrombophilia. ^{3 4} To the best of our knowledge, this very rare concurrent event of SLOS and PC deficiency has yet to be reported in the literature. Here we reported a 6-year-old boy from southeast of Turkey, who had two homozygous mutations of both DHCR7 and PROC genes, detected by using whole exome sequencing (WES). This boy was presented with developmental delay, intellectual disability, microcephaly, lack of speech, seizures, bruxism, repetitive limb movements, self-mutilation, lower extremities contractures, visual impairment, superficial thrombophlebitis, skin scars, and 2 to 3 toe syndactyly.

Case Report

We described here a 6-year-old-boy with homozygous mutations of the DHCR7 and PROC genes, inherited from a consanguineous marriage in Turkey. The parents are clinically normal. The father is 29 years old and the mother is 28 years old. This boy was referred to our department because of developmental delay, lack of speech, seizures, self-mutilation, and dysmorphic features. The patient was the second and last child of the family and his elder brother is clinically healthy. He was born at term by cesarean section due to breech position presentation. His birth weight was 3,100 g (10–25th percentile) and height was 48 cm (10–25th percentile). Occipitofrontal circumference (OFC) was not recorded. When the purpura fulminans developed in the neonatal period, the patient is diagnosed with PC deficiency because of decreased activity (< 10%) by laboratory analysis. Gene mutation study was not performed at that time. At the age of 6 years, his weight was 16.5 kg (3–10th percentile) and her length was 117 cm (50–75th percentile). Clinical examination showed developmental delay, lack of speech, hypertonicity on the lower extremities, involuntary movements, low set ears, long philtrum, ptosis, micropenis, 2 to 3 toe syndactyly, groaning, bruxism, and widespread skin scars ( Fig. 1 ). Abdominal computed tomography (CT) examination showed a marked increase in liver size and a wide hepatic vein. No other anomaly was detected on echocardiography. Biochemical testing of liver, thyroid, and kidney function was normal. The prothrombin time and international normalized ratio (PT-INR) level was 2.55. Total cholesterol was 132 mg/dL. The 7-dehydrocholesterol reductase level was 10 μmol/L (normal < 5 μmol/L). Electroencephalography (EEG) showed epileptic changes. Brain magnetic resonance (MR) imaging demonstrated left lateral ventricle posterior horns were prominent and presented a cavum septum et vergae variant. Eye examination showed a retinal detachment, right seclusion papillae, and 7 × 7 mm leukoma in the right eye. Intraocular pressure values were within normal limits. Microarray (Affymetrix CytoScan Optima-315k; Thermo Fisher Scientific, MA, United States) result was normal.

Fig. 1.

Patient at 6 years of age shows bilateral epicanthus inversus, thin upper lip, ptosis, low set ears, long philtrum, skin scars, and 2 to 3 toe syndactyly.

Based on the clinical and imaging information provided, the preliminary diagnosis was compatible with SLOS, although cholesterol level was within normal limits and 7-dehydrocholesterol reductase was at the borderline. Therefore, WES analysis (Nextera Rapid Capture Exome; Illumina, CA, United States) was ordered, because the patient was suspected to have other gene mutations which may cause a separate syndrome. DNA was extracted from venous blood lymphocytes with the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche, Switzerland). We analyzed variants affecting coding areas and ± 20 splicing regions. The 97.88% of target regions had a coverage depth of more than 10-fold. The analysis showed homozygous variant c.418T > C p. (Cys140Arg) in PROC and homozygous c.57dupC p.(Asn20Glnfs*2) in the DHCR7 . To date, this DHCR7 variant is not described in the Exome Aggregation Consortium (ExAC), 1000 genomes, gnomAD v2.1, Greater Middle Eastern Variome or the NHLBI GO Exome Sequencing Project (ESP). This mutation created a shift in the reading frame starting at codon 20. The new reading frame ends in a stop codon 1 position downstream. It is classified as likely pathogenic (class 2) according to the recommendations of ACMG. In addition, a homozygous variant in the PROC gene was also found. According to HGMD (Human Gene Mutation Database) professional 2018.4, this variant has been previously described as disease-causing for PC deficiency syndrome in one patient. Regarding the mutation detected in the PROC gene, there was no data entry in these databases. Variants confirmed by conventional Sanger's sequencing method and analyzed using SeqScape Software 3 (NextGENe Viewer; SoftGenetics, United States; Fig. 2 ). Based on these findings, a diagnosis of SLOS and PC deficiency was made. Parents and other sibling were all heterozygous for these two changes.

Fig. 2.

Sequencing electropherograms of DHCR7 in the patient.

Discussion

The prevalence of SLOS at birth is estimated to be approximately 1:20,000 to 1:40,000 in live births. The prevalence of severe PC deficiency is about 1 in 500,000–750,000 people in the general population. The treatment of these two conditions would be replacement therapy. In both diseases, the symptoms can be observed at different ages with various severities. ^{2 3 4} According to HGMD Professional 2018, 4,218 mutations related to DHCR7 and 391 mutations related to PROC have been reported. This case report demonstrated an unusual situation with two autosomal recessive disorders. Inherited PC deficiency is one of the rare causes of hereditary thrombophilia, and this rare disorder should be listed in the differential diagnosis especially in the neonatal period presented with purpura fulminans. PC gene, located at chromosome 2q14.3, includes eight exons. Approximately two-thirds of the mutations in the PROC are single nucleotide point mutations. ^{5 6} The identified mutation in PROC exon 5 belongs to the epidermal growth factor-like 2 domain area and is related in a disulfide bridge with Cys151 that binds a β -hairpin component with a β -fiber. The mutation found in this patient likely led to misfolding of the domain or improper S–S bridge during the folding stage. ⁶

SLOS is an autosomal recessive disorder of cholesterol metabolism. Biochemical diagnosis of SLOS is made by steroid measurement. 3 β -hydroxycholesterol Δ7-reductase deficiency leads to a decrease of cholesterol levels and the increase of the 7-dehydrocholesterol (7-DHC) levels. ^{7 8} Although serum concentration of cholesterol is usually low, and it may be within the normal range which could occur in approximately 10% of affected individuals with SLOS. Furthermore, borderline level of 7-DHC was reported in a few cases. All of these patients had very mild SLOS symptoms. ⁹ In contrast, our patient had a normal cholesterol level and the 7-DHC level was at the borderline. Usually this value is higher than 200 μmol/L in most SLOS patients. This finding revealed that even if the cholesterol and 7-DHC levels are normal or borderline, for any cases with clinical suspicion of SLOS, mutation analysis is highly recommended. According to the HGMD, this identified mutation is a small insertional mutation of this gene with only four other insertions being reported. The majority of the previously identified mutations were missense.

Conclusion

In conclusion, we identified the known homozygous mutation in the PROC and a novel homozygous mutation of DHCR7 in a Turkish boy. Besides the known characteristics of SLOS and PC deficiency, we considered that bruxism and lack of speech are part of the clinical presentations in one of these two syndromes. This report also demonstrated (1) the great value of WES in differential diagnosis for complex cases with more than one genetic disorder and (2) the importance of a thorough clinical examination when multiple dysmorphic features were presented. It also should be kept in mind that the coexistence of two autosomal recessive diseases could occur in closely-related consanguineous marriages. ¹⁰

Disclosures

All authors report no conflict of interest related to this manuscript. This research was managed in accordance with the declaration of Helsinki. The patient’s parent gave his informed consent for the molecular genetic analysis of the patient, the publication of patient data and photos.