Identification of a recurrent frameshift mutation at the LDLR exon 14 (c.2027delG, p.(G676Afs*33)) causing familial hypercholesterolemia in Saudi Arab homozygous children.

Familial hypercholesterolemia (FH) is an autosomal dominant disease, predominantly caused by variants in the low-density lipoprotein (LDL) receptor gene (LDLR). Herein, we describe genetic analysis of severely affected homozygous FH patients who were mostly resistant to statin therapy and were managed on an apheresis program. We identified a recurrent frameshift mutation p.(G676Afs*33) in exon 14 of the LDLR gene in 9 probands and their relatives in an apparently unrelated Saudi families. We also describe a three dimensional homology model of the LDL receptor protein (LDLR) structure and examine the consequence of the frameshift mutation p.(G676Afs*33), as this could affect the LDLR structure in a region involved in dimer formation, and protein stability. This finding of a recurrent mutation causing FH in the Saudi population could serve to develop a rapid genetic screening procedure for FH, and the 3D-structure analysis of the mutant LDLR, may provide tools to develop a mechanistic model of the LDLR function.

A CYP11A1 homozygous exonic variant inducing an alternative splicing, frameshift and truncation in a family with congenital adrenal hyperplasia.

Background: Congenital adrenal hyperplasia (CAH) is a heterogeneous group of adrenal steroidogenesis disorders with variable degrees of glucocorticoid, mineralocorticoid and sex steroid deficiencies. CYP11A1 gene encodes the mitochondrial cholesterol side-chain cleavage enzyme (P450scc), which initiates the first reaction in steroidogenesis by converting cholesterol to pregnenolone. Variants in this gene are extremely rare but associated with severe forms of CAH due to its early and critical function in various steroid biosynthesis pathways. Here, we report a CYP11A1 exonic homozygous variant that, although exonic in location, affects splicing by creating an additional aberrant splicing site with frameshift and truncation of the gene. Patients and methods: The proband is a 23-year old 46,XY patient raised as a girl. She was a product of normal pregnancy for first-degree relative parents. Soon after birth, she had vomiting, dehydration, hypotension, hyponatremia and hyperkalemia. She was started on glucocorticoids and mineralocorticoids with prompt recovery. Apart from a chronic need for these medications, her neonatal and childhood history was unremarkable. She sought medical advice at age 19 years for delayed puberty with primary amenorrhea and lack of breast development. On evaluation, she had normal external female genitalia, no breast development, undescended testes and absent uterus and ovaries. Her hormonal evaluation revealed very low estrogen, testosterone, cortisol, aldosterone, 17-hydroxyprogesterone, and androstenedione levels. ACTH, LH, FSH and renin were very high consistent with primary gonadal and adrenal failure. Her parents are healthy first-degree cousins. She has three sisters, all with 46,XX karyotype. One of them is clinically and biochemically normal while the other two sisters have normal female phenotype, normal uterus and ovaries, similar hormonal profile to the proband but different karyotype (46,XX) and absence of undescended testes. gDNA was used for whole exome sequencing (WES). Sanger sequencing was performed to confirm the detected variant and its segregation with the disease. Results: WES identified a homozygous missense variant in CYP11A1 changing the second nucleotide (GCG > GTG) at position 189 in exon 3 and resulting in a change of Alanine to Valine (p.Ala189Val). This variant was confirmed by PCR and Sanger sequencing. It was found in a homozygous form in the proband and her two affected sisters and in a heterozygous form in the unaffected sister. In-silico analysis predicted this variant to create a new splicing site with frameshift and truncation of the gene transcript. This was confirmed by isolation of RNA, cDNA synthesis, gel electrophoresis and sequencing. Conclusion: We describe a family with a very rare form of CAH due to a CYP11A1 variant leading to creation of a new splice site, frameshift and premature truncation of the protein.

25-Hydroxylase vitamin D deficiency in 27 Saudi Arabian subjects: a clinical and molecular report on CYP2R1 mutations.

Vitamin D deficiency remains a major cause of rickets worldwide. Nutritional factors are the major cause and less commonly, inheritance causes. Recently, CYP2R1 has been reported as a major factor for 25-hydroxylation contributing to the inherited forms of vitamin D deficiency. We conducted a prospective cohort study at King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia, to review cases with 25-hydroxylase deficiency and describe their clinical, biochemical, and molecular genetic features. We analyzed 27 patients from nine different families who presented with low 25-OH vitamin D and not responding to usual treatment. Genetic testing identified two mutations: c.367+1G>A (12/27 patients) and c.768dupT (15/27 patients), where 18 patients were homozygous for their identified mutation and 9 patients were heterozygous. Both groups had similar clinical manifestations ranging in severity, but none of the patients with the heterozygous mutation had hypocalcemic manifestations. Thirteen out of 18 homozygous patients and all the heterozygous patients responded to high doses of vitamin D treatment, but they regressed after decreasing the dose, requiring lifelong therapy. Five out of 18 homozygous patients required calcitriol to improve their biochemical data, whereas none of the heterozygous patients and patients who carried the c.367+1G>A mutation required calcitriol treatment. To date, this is the largest cohort series analyzing CYP2R1-related 25-hydroxylase deficiency worldwide, supporting its major role in 25-hydroxylation of vitamin D. It is suggested that a higher percentage of CYP2R1 mutations might be found in the Saudi population. We believe that our study will help in the diagnosis, treatment, and prevention of similar cases in the future.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR): clinical heterogeneity and long-term efficacious management of eight patients from four unrelated Arab families with a loss of function VDR mutation.

BACKGROUND: Vitamin D regulates the concentrations of calcium and phosphate in blood and promotes the growth and remodeling of bones. The circulating active form of vitamin D, 1,25-dihydroxyvitamin D, binds to the vitamin D receptor (VDR), which heterodimerizes with the retinoid X receptor to regulate the expression of target genes. Inactivating mutations in the VDR gene cause hereditary vitamin D-resistant rickets (HVDRR), a rare disorder characterized by an early onset of rickets, growth retardation, skeletal deformities, hypocalcemia, hypophosphatemia and secondary hyperparathyroidism, and in some cases alopecia. METHODS: We describe eight new HVDRR patients from four unrelated consanguineous families. The VDR gene was sequenced to identify mutations. The management of patients over a period of up to 11 years following the initial diagnosis is assessed. RESULTS: Although all patients exhibit main features of HVDRR and carry the same c.885C>A (p.Y295*) loss of function mutation in the VDR gene, there was heterogeneity of the manifestations of HVDRR-associated phenotypes and developmental milestones. These eight patients were successfully treated over a period of 11 years. All clinical symptoms were improved except alopecia. CONCLUSIONS: The study concludes that VDR sequencing and laboratory tests are essential to confirm HVDRR and to assess the effectiveness of the treatment.

Compound heterozygous LDLR variant in severely affected familial hypercholesterolemia patient.

Familial hypercholesterolemia (FH) is most commonly caused by mutations in the LDL receptor (LDLR), which is responsible for hepatic clearance of LDL from the blood circulation. We described a severely affected FH proband and their first-degree blood relatives; the proband was resistant to statin therapy and was managed on an LDL apheresis program. In order to find the causative genetic variant in this family, direct exon sequencing of the LDLR, APOB and PCSK9 genes was performed. We identified a compound heterozygous mutation in the proband with missense p.(W577C) and frameshift p.(G676Afs33) variants at exons 12 and 14 of the LDLR gene respectively. DNA sequencing of LDLR gene from the parents demonstrated that the missense variant was inherited from the mother and frameshift variant was inherited from the father. The frameshift variant resulted in a stop signal 33 codons downstream of the deletion, which most likely led to a truncated protein that lacks important functional domains, including the trans-membrane domain and the cytoplasmic tail domain. The missense variant is also predicted to be likely pathogenic and affect EGF-precursor homology domain of the LDLR protein. The segregation pattern of the variants was consistent with the lipid profile, suggesting a more severe FH phenotype when the variants are in the compound heterozygous state. The finding of a compound heterozygous mutation causing severe FH phenotype is important for the genotype-phenotype correlation and also enlarges the spectrum of FH-causative LDLR variants in the Arab population, including the Saudi population.

Next generation sequencing to identify novel genetic variants causative of autosomal dominant familial hypercholesterolemia associated with increased risk of coronary heart disease.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is an autosomal dominant disease, caused by variants in Ldlr, ApoB or Pcsk9, which results in high levels of LDL-cholesterol (LDL-C) leading to early coronary heart disease. Sequencing whole genome for screening variants for FH are not suitable due to high cost. Hence, in this study we performed targeted customized sequencing of FH 12 genes (Ldlr, ApoB, Pcsk9, Abca1, Apoa2, Apoc3, Apon2, Arh, Ldlrap1, Apoc2, ApoE, and Lpl) that have been implicated in the homozygous phenotype of a proband pedigree to identify candidate variants by NGS Ion torrent PGM. Only three genes (Ldlr, ApoB, and Pcsk9) were found to be highly associated with FH based on the variant rate. The results showed that seven deleterious variants in Ldlr, ApoB, and Pcsk9 genes were pathological and were clinically significant based on predictions identified by SIFT and PolyPhen. Targeted customized sequencing is an efficient technique for screening variants among targeted FH genes. Final validation of seven deleterious variants conducted by capillary resulted to only one novel variant in Ldlr gene that was found in exon 14 (c.2026delG, p. Gly676fs). The variant found in Ldlr gene was a novel heterozygous variant derived from a male in the proband.

Identification of a novel nonsense variant c.1332dup, p.(D445*) in the LDLR gene that causes familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disease predominantly caused by a mutation in the low-density lipoprotein receptor (LDLR) gene. Here, we describe two severely affected FH patients who were resistant to statin therapy and were managed on an apheresis program. We identified a novel duplication variant c.1332dup, p.(D445*) at exon 9 and a known silent variant c.1413A>G, p.(=), rs5930, NM_001195798.1 at exon 10 of the LDLR gene in both patients.