Discussion
Vitamin D has two main forms, ergocalciferol (vitamin D2) and
cholecalciferol (vitamin D3). The source of ergocalciferol is food such
as liver oil, milk, fish and eggs, while the source of cholecalciferol
is the skin under the influence of ultraviolet B. The next step is for
the synthesized vitamin D to be transported to the liver via its carrier
protein. Then it is modified by 25-hydroxylase, where hydroxylation
first occurs. Synthesized calcidiol or calcifediol is absorbed by
proximal convoluted tubule (PCT) receptors such as megalin and cubilin
by endocytosis(6). Calcitriol or
1,25-dihydroxy vitamin D (the active form of vitamin D) is synthesized
by 1 alpha-hydroxylase in the kidney, where the secondary hydroxylation
takes place(7). CYP2R1 and CYP27B1 encode
hydroxylating enzymes of primary (calcidiol) and secondary product
(calcitriol), respectively(6,
7).
Calcitriol binds to its receptor on the surface of enterocytes and
subsequently increases expression of the Transient Receptor Potential
Cation Channel Subfamily V Member 6 (TRPV6) calcium channels, resulting
in increased calcium absorption. Balance of bone metabolism is
maintained by intericate interaction between PTH, fibroblast growth
factor-23 (FGF-23), and 1,25 dihydroxy vitamin D hormones. This
interaction helps us to understand the correlation between these and the
mechanisms of skeletal demineralization in rickets patients. Calcitriol
synthesis in the kidney is increased by factors such as PTH,
hypocalcemia, and hypophosphatemia. These two hormones (PTH and
calcitriol) increase FGF-23 level. FGF-23 is a hormone produced by
osteocytes and is regulated by two major bone proteins, including
Phosphate Regulating Endopeptidase Homolog X-Linked(PHEX) and Dentin
matrix protein 1(DMP1). Decreased expression of these two proteins
promotes osteomalacia. This hormone lowers the calcitriol and PTH
levels. FGF-23 plays a role in renal excretion of phosphate by acting on
its (FGF) receptor in the kidney. This hormone acts through a tubular
membrane protein called klotho downregulating the major sodium /
phosphate co-transporters in the proximal tubules, including NaPi-2a and
NaPi-2c, resulting in increased
phosphaturia(8,
9).
The finding of bone deformities due to disruption of bone mineralization
in the area of the growth plate is a hallmark of rickets, the most
common form of which is nutritional
related(10). In general, impaired vitamin
D metabolism is associated with four types of rickets, including VDDR1A,
VDDR1B, VDDR2A or VDDR and VDDR2B, and is characterized by defects in
proper expression of CYP27B1, CYP2R1, vitamin D receptors, and protein
related to vitamin D receptor function,
respectively(1,
11). Pseudo-VDDR (PDDR) or VDDR1
presented with hypotonia, hypoplasia of dental enamel, growth
retardation, weakness, and developmental delay at the age of 6-24
months. It can also manifest as a seizure in the first year of life
(12). Alterations in the VDDRIA gene can
be compound heterozygous or homozygous, both of which are inherited in
an autosomal recessive manner(13).
Referring to the Human Gene Mutation Database (HGMD), as of January
2020, 81 different CYP27B1 gene mutations associated with VDDR-IA
disease have been reported (14).
Mutations in this gene are more commonly associated with missense and
nonsense forms, although deletions, splice site alterations,
duplications, and insertions have also been
reported(15).The location of CYP27B1 gene
is on the long arm of chromosome 12. The locus from 12q13.1 - q13.3 is
5Kb in size and contains 9 exons(16) and
8 introns translated into 508 amino acids
(17).
Clinical findings of rickets (vitamin D deficiency form) can be divided
into two categories: skeletal and extra skeletal; of the skeletal
manifestations, bone changes are a typical feature of the disease and
vary according to the site of stress. The areas of greatest bone
involvement are usually areas of high growth, such as the epiphyses of
long bones and costochondral
junctions(18). In toddlers and crawling
infants, bone involvement in the form of genu varum or genu valgum and
forearm deformities are more
predominant(19). Ambiguous presentations
in adolescence, such as headaches and lower extremity pain are called
florid signs, which are uncommon. Craniotabes, widened fontanelles, bone
mass in the frontal region (frontal bossing), delayed growth of teeth,
rachitic rosary (which reflects the severe form of rickets caused by
enlargement of the costochondral region)
(20), looser’s zone, and pathologic
fracture(21) have been observed in these
patients(19,
22). For extra skeletal findings;
irritability, proximal limb weakness,
hypotonia(23), laryngospasm, tetany,
seizure and cardiomyopathy (uncommon) have been
reported(3,
24).
On radiological examination of these patients; the epiphyseal center may
be delayed or in ill-defined, small and osteopenic forms. The
metaphyseal findings in these patients are regarding its expansion.
During the early and advanced stages, changes such as the absence of
crisp line and concave or frayed form, respectively occurred
(25). All epiphyses in rickets are
affected. In particular, the growth plates in area with faster growth,
so radiological findings are more
pronounced(18). The Rickets Severity
Score (RSS) can be used to assess the transformation of metaphysis and
growth plates more accurately. RSS is also a useful measure for
assessing patient response to treatment and disease severity
(26). X-rays are routinely used in the
diagnose of rickets, and the role of magnetic resonance imaging (MRI) in
showing cartilage changes in rickets was recently
reported(27).
As the clinical diagnosis of rickets becomes available to patients,
laboratory tests is help to achieve a rapid diagnosis. In addition to
diagnosis, these assessments serve to reflect the type and stage of
rickets. In patients with bone deformation and epiphysis expansion,
measurement of ALP levels may be an salient finding in confirming
rickets. Monitoring ALP level may be a useful tool for assessing disease
progression and treatment response(18).
Recently, Mukai, M. et al., represented that ALP level had bidirectional
relationship with the degree of genu varum during a study of pediatric
rickets due to vitamin D deficiency with genu varum
(28). A common feature of all types of
rickets is low serum phosphate levels
(29). Indeed, hypophosphatemia in
patients with vitamin D-dependent rickets can be managed by adjusting
calcium to mean normal serum levels and normalizing PTH levels.
Radiographic examination, plasma and urine biochemical concentration,
and kidney ultrasound should be checked regularly.
There is evidence that calcitriol or alfacalcidol can be used for high
levels of ALP and PTH and low serum calcium and phosphorus levels in
patients with VDDR1a (30). There is
evidence that calcitriol or alfacalcidol can be used for high levels of
ALP and PTH and low serum calcium and phosphorus levels in patients with
VDDR1a which clinical and paraclinical findings of patients improved by
a physiological dose of
1,25(OH)2D(31-33). The recommended dose
for an adequate response in these patients is 1–2 µg/day of calcitriol,
and the tolerable calcium intake for these patients is 30–75 mg/kg/day
(5). In addition to initiating treatment
for patients, it is necessary to re-evaluate calcium and phosphorus
(serum-urinary level), ALP, calcidiol, because knowing the values of
these findings are a good guide to adjust the patient’s treatment. The
first expected change after starting treatment is an increase in
phosphate, followed by and increase in serum calcium. Six months after
starting treatment, the patient’s clinical symptoms may have resolved.
Although, the first radiologic changes can be seen from the first
week(34), its improvement occurs 6-8
weeks after the starting treatment(5).
Complications from initiation of treatment with calcitriol include
elevated serum, urinary, and tissue calcium levels, and regular
evaluation for nephrocalcinosis, hypercalcemia, and hypercalciuria is
recommended (5). Urinary calcium/
creatinine ratio can be used to reflect the degree of hypercalciuria in
these patients (34).
Our patient’s mutation was homozygous c.195 + 2T> G in exon
1 (splice site pathogenic form), and its early symptoms were hypotonia,
movement disorders and growth retardation. According to our patient’s
mutation, Fatma Dursun et al. evaluated the genomic DNA of 11 VDDR1A
patients. They found that the c.195 + 2T> G splice donor
site was the most common mutation reported in 7 patients out of 11
participants, 2 of whom were compound heterozygous, 5 were
homozygous(11). For the first time, the
mutation c.195 + 2T> G (homozygous) was reported in two
Turkish cases from the same family(35).
Fatma Dursun et al. reported movement disorders and bowed legs in all
patients with intron-1 mutations. They noted that the height percentage
of 4 patients with intron-1 mutations was less than 2 standard
deviations. In addition, an 11-month-old patient had an episode of
hypocalcemic convulsion(11). Tahir et al.
assessed the genotype and phenotype of 22 VDDR1 cases, in which they
discussed 10 patients with c.195 + 2T> G mutations (splice
donor site). They showed that individuals with intron-1 mutations
exhibited milder symptoms, with the exception of one patient who
presented with hyocalcemic convulsion. No good correlation has been
reported between genotype and phenotype of VDDR1
patients(36). Durmaz et al. evaluated the
genotypic and phenotypic findings of 15 cases of VDDR1 from 4 families.
They observed 4 cases with the c.195 + 2T> G mutation
(homozygous), 2 of whom had severe hypocalcemic convulsion at 4 months
of age and the others showed delayed movement along with mild
hypocalcaemia at the age of 18-19 months. They reported that there was a
good relationship between genotype and phenotype in VDDR1 patients
(35).
Demir et al. analyzed CYP27B1 gene mutations of eight patients and their
seven families. They found that 3 patients had c.195 + 2T>
G mutations (homozygous), 2 patients had movement and growth disorders,
and 1 of them had growth disorder and fractures. They reported that half
of the patients presented with hypocalcemic convulsion. They stated that
intron-1 mutations were associated with the most severe symptoms among
the mutations, thus they did not report a pertinent relationship between
genotype and phenotype in VDDR1
patients(37). Kaygusuz et al evaluated
the correlation between genotype and clinical presentation in 13
patients with VDDR-IA. They argued that the c.195 + 2T> G
mutation is one of the most common mutations. Of the 13 patients, 4
patients had movement disorders and only 1 patient had a hypocalcemic
seizure. They reported that there was a clear association between
genotype and clinical manifestations in VDDR-IA patients, and that
patients with intron-1 c.195 + 2T> G mutations had more
severe symptoms owing to abnormal height standard deviation score (SDS),
so they need higher doses of
calcitriol(14). To analyze the genotype
and clinical manifestations of VDDR-IA patients, Dodamani et al.
designed a retrospective study of 7 VDDR-IA patients and a systematic
review of 165. A systematic evaluation of 165 patients showed that c.195
+ 2T> G mutation was one of the most common mutations and
that mutation was region specific (West Asia, Turkey). All seven
patients had abnormal height SDS, deformity, high PTH and ALP, low
calcium level, and low 1,25 (OH)2D at baseline. Of the 7
patients, 4 had episodes of hypocalcemic seizure. In 165 patients,
seizures and motor-deformity were more common during infancy(11 of 31
infants) and post-infancy,
respectively(38).
The therapeutic dose of calcitriol range in VDDR1 patients is 0.008 -
0.40 μg / kg / day or 0.5 - 2 μg /
day(33). Yunfei Li et al. introduced two
cases of VDDR1 and discussed that both responded to high doses of
calcitriol (case 1: 0.75 μg / day or 0.079 μg / kg / day and case 2: 2
μg / day or 0.18 μg / kg / day)(39). In a
study conducted by Durmaz et al., doses of 0.01–0.1 μg / kg / day
normalized serum levels of phosphate and calcium and improved growth
status in VDDR1 patients (35).
Ningyi Cui et al. examined the CYP27B1 gene in 8 Chinese patients with
PDDR and obtained therapeutic response with doses of 0.5–1.0 μg / day
of calcitriol (40). Valentina Donghi et
al. regarded proper calcium diet with regimen of 25-30 ng / kg / day
Calcitriol, which gradually raised up to 50 ng / kg / day for VDDR1
cases. Optimal improvement of clinical and paraclinical findings of
VDDR1 patients was achieved after 12 weeks of treatment
(41). T. Edouard et al. evaluated
clinical findings in PDDR patients during calcitriol treatment. They
initially took 0.5 µg of calcitriol twice daily, but after 3 months of
acceptable feedback, they reduced the therapeutic dose to 0.50 µg / d.
The calcitriol dose was then changed to 0.25 µg / d (0.1-1.0 µg) and
0.25 µg / d (0.1-0.5 µg) after 1 year and 2 years,
respectively(42). Kaygusuz et al. noted
in their study that the intended dose of calcitriol was 41.01 ± 7.59 ng
/ kg / day depending on the severity of symptoms and hypocalcemia.
Clinical and paraclinical improvements with calcitriol regimen were
observed in all patients (calcium level improved after 1-3 months, PTH
and ALP levels improved after 3-12
months)(14). Dodamani et al. received
optimal therapeutic feedback with a diet of calcium (30–75 mg / kg /
day) and calcitriol (10–20 ng / kg / day). At the onset of radiographic
changes, the calcitriol dose modified to 0.25-0.5 μg / day. They
concluded that physiological doses of calcitriol (0.25-0.75 μg / day)
could improve the clinical and laboratory profile of patients
(38). Dhull et al. considered doses of 30
ng/kg/day and 2 mmols/kg/day for calcitriol and phosphate, respectively,
for both patients. Then, after positive feedback, the calcitriol dosage
was modified; it was set to 20 ng/kg/day in the first case and reduced
to 10 ng/kg/ day in the second case. Also, over the course of treatment,
phosphate supplementation was reduced and then
discontinued(43).
It is clear from the therapeutic doses in these studies is that some
patients respond to high doses of calcitriol and others respond to low
doses of calcitriol, but the dose chosen should be based on the
individual patient. Please note that the decision should be made on a
case-by-case basis. However, complications such as hypercalciuria and
nephrocalcinosis should be considered.
A typical finding in patients with VDDR1 is decrease serum
1,25-OH2D levels, although normal
1,25-OH2D levels have been reported at disease
onset(37,
44). Recently, Nishikawa et al proposed
that enzymes other than CYP27B1 are involved in 25-OHD catalysis. They
showed that in knockout mice (CYP27B1 enzyme), 1,25-OH2D
is recovered from 25-OHD by the CYP27A1
enzyme(45). In contrast to our patient,
who had low 1,25-OH2D levels, Giannakopoulos et
al.(46) and Wang et
al.(47) reported 1 and 2 cases,
respectively, with normal 1,25-OH2D levels. In a study
by Dursun et al., 8 of the 11 patients had normal or above normal levels
of 1,25-OH2D. Of the 5 patients with relatively high 1,
25-OH2D levels, all five received vitamin D treatment due to
misdiagnosis of nutritional rickets. Of the 3 patients with normal
1,25-OH2D levels, 1 patient was treated with phosphate
and calcitriol for misdiagnosis of hypophosphatemic
rickets(11). Dhull et al. reported 2
cases with mutations in the CYP27B1 gene and both impaired movement and
growth. Their baseline laboratory results demonstrated a normal reading
of 1,25 (OH)2D(43).