Carvalho et al, NGF and insensitivity to pain.
Supplemental data
Clinical data.
Detailed phenotype information on all five affected siblings.
Case 1
The main complaint of this 12.5 year old girl is lack of sensitivity to pain, both somatic and visceral. She also has no sense of temperature and cannot distinguish between warm and cold water. She never blushes. She does not sweat and the parents keep her out of the heat because otherwise she develops high temperature.
She has a normal sense of touch, feels pressure and laughs when tickled. Her sense of smell and taste seem to be normal, she shows love and affection in a normal way. She has tears.
She is mentally mildly delayed. Indeed she is only in the 4th class of primary school and gets special additional tutoring. She is very active and does not get tired easily. Her gross and fine motor developments are normal. She sees and hears well. She loses her teeth very easily. Most of her molar teeth are gone.
The pregnancy was complicated by premature rupture of membranes and meconium stained liquor. C-section was proposed but refused. Apgar scores were 4 at 1 minute, 7 at 5 minutes and 9 at 10 minutes. Her birth weight was 3.04 kg, length 52 cm, and HC 33.5cm
The parents first noticed that she had not sense of pain around the age of 6 months when she started to bite her lips till they bled. By the age of 1 year they noticed that she would burn herself without showing any sign of pain. She used to bite her tongue and fingers till she was old enough to understand; currently she no longer harms herself voluntarily.
She had several admissions to hospital because of deep cuts and or bone fractures which healed well without complications. She has never had other serious infections suggestive of immunodeficiency. At the age of 11 years she developed swelling of the left knee without a clear history of trauma. There was only mild effusion and no other signs of infection. X-Ray was normal and she recovered spontaneously.
Physical examination at the age of 12.5 years showed a weight of 29.6 kg (< P3); height of 137.2 cm (< P3); BMI 15.7 P10-25 (mother’s height was 152.3 cm and the father is apparently tall).
She appeared in a good general condition and without dysmorphic features apart from retracted or “sunken” mouth. The edge of the tongue was scarred and looked a bit shorter than expected. Her teeth were of poor quality, irregular and crowded. She had lost most of her molars but still had her incisors. The uvula appeared bilobar. The distal phalanx of her right thumb was shortened and had a small thickened nail, due to a previous injury. The distal phalanx of her left index was also lost. She had increased vein pattern of arms and legs. Heart, lung and abdominal examination were normal. There were no signs of puberty.
Investigations including complete blood count and immunoglobins were normal.
(WBC 6.9; Ig A 2.15 g/L; Ig G 9.59 g/L; Ig M 0.98 g/L (all these results are normal))
Umbilical cord fell off at normal time.
She is no more annoyed by bright light or loud noise than others. Does not feel that ice cream is cold, does not feel spicy taste as hot.
Reaction to insect bites is normal concerning redness and swelling.
Superficial wounds, grazes and cuts, take an abnormally long time to heal.
Case 2
This 11.5 year old boy is the first of twins. He has a brother with the same symptomatology of profound bilateral neuro-sensorial hearing loss and lack of speech in addition to the familial congenital insensitivity to pain. He was the product of uncomplicated twin pregnancy and was delivered by LSC. Apgar scores were 1 at 1 minute, 6 at 5 minutes, 8 at 10 minutes.
His birth weight was 3.23 kg, length 54 cm, and HC 35 cm. During the resuscitation he was wrapped up and put under the radiant warmer. His temperature went up to 39.5 and stayed high for several hours – maybe due to lack of sweating.
Mother noticed no sense of pain by the age 2 months. He started biting his lips and tongue from the moment he got teeth. At the age of 5.5 months he was admitted for pneumonia and was found to have tissue loss of the distal phalanx of the right second finger. Bone was protruding and possible osteomyelitis was diagnosed. At the age of 6 years he had osteomyelitis of the distal phalanx of the right big toe needing curettage. At the age of 8 years he developed intra-abdominal hematoma around the duodenum. It was assumed that this was caused by a blunt abdominal injury, although the family denied any history of trauma.
The child had recurrent septic arthritis of the right knee since the age of 8.5 years. At the age 10.5 he was admitted because of recurrence of the septic arthritis of the right knee and an abscess of the right thigh with beginning osteomyelitis. MRI showed supracondylar fracture of the right distal femur with chronic instability of the distal femoral epiphysis.
His early motor developmental milestones were normal but he attends a school for children with special needs and is currently in the 2nd grade. It is very difficult to assess his mental development because of his deafness and lack of teaching of sign language. However, he is probably mildly retarded.
Like his sister he does not sweat and develops high temperature when goes out in the sun.
Physical examination revealed a weight of 26.5 kg (< P3); height of 133.2 cm (< P3); BMI 14.9 (P5). He had no dysmorphic features apart from a deformed nose due to a previous fracture. The edge of the tongue appeared scarred and looked a bit shorter than expected. The teeth were irregular and crowded and the lower incisors were “bent back” a bit but there was no loss of teeth. The uvula appeared bilobar. There were many old skin scars all over the body. The distal phalanges of the first 3 fingers of the right hand as well as the distal phalanx of the right big toe were shortened with only a rudimentary nail left. The right knee was double the size of the left one but with a normal range of motion. There were no signs of puberty.
Investigations including complete blood count and immunoglobines were normal
(WBC 11 Neutrophil 70% , Ig A 3.47 g/L; Ig G 14 g/L; Ig M 1.04 g/L all normal)
He is not more annoyed by bright light than others. Does not feel that ice cream is cold, does not feel spicy taste.
Umbilical cord fell off at normal time.
Reaction to insect bites is normal concerning redness and swelling.
Mother feels that his wounds take an abnormally long time to heal.
Case 3
This 11.5 year boy is the second of the twins whose brother is discussed above. Like his brother he has profound bilateral neuro-sensorial hearing loss and lack of speech in addition to the congenital insensitivity to pain.
He was the product of normal pregnancy and LCS. Apgar scores were 7 at 1 minute, 9 at 5 minutes, 9 at 10 minutes. His birth weight was 2.74 kg, length 51 cm and HC 33.5 When put under the radiant warmer his temperature went up to 39.2 but normalized within 3 hours – a sepsis screen remained negative.
Mother noted inability to feel pain at the age of 2 months. He started biting his tongue, lips and fingers from the moment he got teeth.
Over the years he had several fractures involving different bones. At the age of 1.5 years he got a cut wound of the right 4th finger which got badly infected. On exploration the distal epiphysis of middle phalanx was found to be necrotic and had to be removed.
At the age of 9.5 years he ruptured the left eye globe due to trauma by pen. He has a severe residual scar of the cornea.
At the age of 11.5 years he developed septic arthritis of the left ankle with acute osteomyelitis.
His early motor developmental milestones were normal. His mental development is similar to his brother’s and he also attends a school for children with special needs.
Examination at the age of 11.5 years revealed a weight of 25.6 kg (< P3); height of 135.2 cm (< P3) and BMI 14.01 (< P3). There was corneal scar of the left eye. The nose appeared deformed due to a previous fracture. The edge of the tongue was scarred and looked a bit shorter than expected but overall the tongue and the lips look less scarred than his twin brother’s. The teeth were irregular and crowded. The uvula appeared broad. There were a lot of old scars all over the body. The distal phalanges of second finger of right hand as well left thumb were shortened with only a rudimentary nail left. The last phalanx of right 3rd finger was fused with the middle phalanx but without full alignment. There were no signs of puberty.
Investigations including complete blood count and immunoglobins were normal.
(WBC 10.9, Ig A 3.6 g/L; Ig G 13.83 g/L; Ig M 1.0 g/L all normal)
Case 4
This 5 year old girl had a normal perinatal history. Her birth weight was 3.225 kg, length 54 cm and HC 34 cm. Apgar scores were 8 at 1 minute, 9 at 5 minutes and 10 at 10 minutes. There were no neonatal problems. She had bilateral dysplastic hips which responded well to conservative treatment.
Like her other sibs she does not feel pain. She still bites her tongue, lips and fingers. She has lost all her teeth. Over the years she had several burns and ulcers. The parents do not feel that she is mentally normal. Umbilical cord fell off at normal time.
She is no more annoyed by bright light or loud noise than others. Does not feel that ice cream is cold, does not feel spicy taste as hot.
Reaction to insect bites is normal concerning redness and swelling.
Mother feels that her wounds take an abnormally long time to heal.
Examination revealed a weight of 16 kg (P10-25), length of 106.4 cm (P 10-25 and weight for stature at P10-25. She looked well and physical development had been normal, but she was exhibiting the same cognitive pattern as her affected siblings and appeared also to be mildly mentally retarded. She had only a few teeth, most of them with caries. The uvula was bilobar. At the time we saw her, she had lost the nails of two fingers but all her phalanges were intact. The feet were intact.
All investigations were normal (WBC 6.8 neutrophil 34.3, IgA 2.06 (slightly high) g/L; Ig G 10.7 g/L; Ig M 1.0 g/L all normal)
Case 5
This 23 months’ old girl was the product of normal pregnancy and delivery. Her birth weight was 3.185 kg, length 52 cm and HC 34 cm.
She started walking at the age of 1 year and now, at the age of 23 months, uses 5 to 6 words. She still bites her tongue, lips and fingers. She has lost many of her teeth.
Umbilical cord fell off at normal time.
She is no more annoyed by bright light or loud noise than others. Does not feel that ice cream is cold, does not feel spicy taste as hot.
Reaction to insect bites is normal concerning redness and swelling.
Mother feels that her wounds take an abnormally long time to heal.
Examination revealed a weight of 10.8 kg (P10-25), length of 82.6 cm (P 25-50 ) and weight for stature at P 25-50. She looked healthy and development to 23 months was normal. The front teeth were missing and there was caries of the premolars.
The first and second distal phalanges of the left hand and the second and third distal phalanges of the right hand were swollen, with nails that appeared rudimentary, doubtlessly the results of self-inflicted bites. There was a severe healing burn of about 4cm diameter on the right forearm.
Investigations were normal (WBC 8.8 29% , Ig A 0.48 g/L; Ig G 5.62 g/L; Ig M 0.73 g/L)l
Methods
Clinical studies. The family was ascertained through a local Clinical Genetics service after they sought a diagnosis and explanation for their children’s difficulties. Research Ethics approval for this work was gained in United Arab Emirates and the United Kingdom; however nerve biopsy and formal assessments of pain were not considered justifiable.
Genotyping and mutation detection.
DNA was extracted from all family members using standard protocols. Autozygosity mapping was performed on three affected family members using the Affymetrix 250K Nsp1 SNP chip following the manufacturer’s protocol. Data was analysed with GTYPE, GCOS and CNAG. Concordant homozygous regions were visualised using the Human Genome Browser with flanking heterozygous SNPs to delineate regions of interest. Candidate genes were sequenced using patient genomic DNA. Specific primers were designed using the Primer3 program (Steve Rozen and Helen J. Skaletsky, 2000) to include all exons and their splice sites, the stop codon and polyadenylation signal. Standard bidirectional sequencing protocols using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) were used as per the manufacturer protocol. The sequence obtained was compared to the published reference sequence and mutations were identified.
Cell culture and transfection
Rat pheochromocytoma PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 5% Foetal Calf Serum, 10% Horse Serum, 2mM L-glutamine, 100μg/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37˚C in a humidified atmosphere with 10% CO2. Simian kidney COS-7 cells were maintained in complete media (DMEM supplemented with 10% Foetal Calf Serum, 2mM L-glutamine, 100μg/ml penicillin and 100 μg/ml streptomycin). Cells were maintained at 37˚C in a humidified atmosphere with 5% CO2. Both cell types were transiently transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturers’ recommendations. 24 hours before transfection, cells were plated at a density of 25 000 - 15 000 cells/ cm2. Transfections were carried out in Opti-MEM Reduced Serum Media (Invitrogen). After 4-6 hours the media was replaced with complete media.
Transfection plasmid construction