Ental retardation Contractures Early infantile hypotonia Spastic tetraplegia Hypertonia Hyperreflexia Babinski sign Extensor plantar reflex MedChemExpress 61177-45-5 Epilepsy Deambulation Normal speech Sphincter control Eye evaluation Hearing evaluation Neuroimaging (CT) Loss of acquired function Purposeful hand use Pseudobulbar signsP1 AP4E1 F Infancy 3 46 cm (22SD) +++ ?+ Lower extremities only ?????Never achieved Never acquired Absent Slight 
myopia and astigmatism Transmission deafness on left side, normal on right side Atrophy of the inferior vermis with cortical atrophy ++ ++ Drooling+++, stereotypic laughter+++, jaw jerk++, gag reflex++P2 AP4E1 F Infancy 3 45.5 cm (22.5SD) +++ ?+ Lower extremities only ?????Never achieved Never acquired Absent Slight astigmatism ?Atrophy of the inferior vermis with cortical atrophy ++ ++ Drooling+++, stereotypic laughter+++, jaw jerk++, gag reflex++doi:10.1371/journal.pone.0058286.tPatients and Methods Case ReportsThe two patients (P1 and P2) are identical twins born to firstcousin Moroccan parents (Figure 1A). No complications were observed during the pregnancy, but neonatal asphyxiation was noted during the 117793 delivery. When examined at the age of three years, the twins displayed marked developmental retardation, including microcephaly, an inability to walk unaided, abnormal speech and abnormal circadian development. They kept smiling or laughing for no obvious reason. Abnormal drooling was also observed. Both patients had muscular hypotonia. No seizure or involuntary movement was observed in either of the patients. Both patients presented discreet but remarkable facial gestalt with a prominent, bulbous nose, a wide mouth and coarse features. Neurological examination revealed spastic paraplegia of the lower extremities. Denver II assessments of both children demonstrated a significant delay in the acquisition of major skills, such as motor and adaptive skills, language and social skills, at the age of three. Both patients were of short stature and had a low body weight. Their clinical features are summarized in detail in Table 1. Electroencephalography (EEG) revealed a diffusive slow wave (theta and delta wave). Based on these findings, both twins were diagnosed with type I complex HSP. In addition to neurologic problems, both patients presented unilaterally enlarged and inflammatory axillary lymph nodes at nine months of age. Both had been vaccinated with BCG (a live attenuated strain of Mycobacterium bovis), which was injected into theshoulder a few days after birth. The enlarged lymph nodes were removed surgically. Biopsy confirmed the presence of acid-fast bacilli, consistent with mycobacterial infection. Neither of the patients had presented any other episode 
of mycobacterial infection by the time of clinical evaluation at three years of age.Ethics StatementThis study was conducted in accordance with the Helsinki Declaration, with written informed consent obtained from the parents of P1 and P2 and the other healthy individuals involved. Approval for this study was obtained from the French IRB (Comite ?de protection des personnes or CPP), INSERM and the Rockefeller IRB.Epstein-Barr Virus (EBV) Transformation of B Lymphocytes (EBV-B Cells) and Cell CulturePBMC were isolated from 10 ml of peripheral blood on a Ficoll gradient and were suspended in 4 ml of RPMI1640 supplemented with 20 fetal calf serum (FCS) and 0.2 mg/ml cyclosporine A. We then added 1 ml of EBV medium from the B95.8 cell line [28]. We replaced half the.Ental retardation Contractures Early infantile hypotonia Spastic tetraplegia Hypertonia Hyperreflexia Babinski sign Extensor plantar reflex Epilepsy Deambulation Normal speech Sphincter control Eye evaluation Hearing evaluation Neuroimaging (CT) Loss of acquired function Purposeful hand use Pseudobulbar signsP1 AP4E1 F Infancy 3 46 cm (22SD) +++ ?+ Lower extremities only ?????Never achieved Never acquired Absent Slight myopia and astigmatism Transmission deafness on left side, normal on right side Atrophy of the inferior vermis with cortical atrophy ++ ++ Drooling+++, stereotypic laughter+++, jaw jerk++, gag reflex++P2 AP4E1 F Infancy 3 45.5 cm (22.5SD) +++ ?+ Lower extremities only ?????Never achieved Never acquired Absent Slight astigmatism ?Atrophy of the inferior vermis with cortical atrophy ++ ++ Drooling+++, stereotypic laughter+++, jaw jerk++, gag reflex++doi:10.1371/journal.pone.0058286.tPatients and Methods Case ReportsThe two patients (P1 and P2) are identical twins born to firstcousin Moroccan parents (Figure 1A). No complications were observed during the pregnancy, but neonatal asphyxiation was noted during the delivery. When examined at the age of three years, the twins displayed marked developmental retardation, including microcephaly, an inability to walk unaided, abnormal speech and abnormal circadian development. They kept smiling or laughing for no obvious reason. Abnormal drooling was also observed. Both patients had muscular hypotonia. No seizure or involuntary movement was observed in either of the patients. Both patients presented discreet but remarkable facial gestalt with a prominent, bulbous nose, a wide mouth and coarse features. Neurological examination revealed spastic paraplegia of the lower extremities. Denver II assessments of both children demonstrated a significant delay in the acquisition of major skills, such as motor and adaptive skills, language and social skills, at the age of three. Both patients were of short stature and had a low body weight. Their clinical features are summarized in detail in Table 1. Electroencephalography (EEG) revealed a diffusive slow wave (theta and delta wave). Based on these findings, both twins were diagnosed with type I complex HSP. In addition to neurologic problems, both patients presented unilaterally enlarged and inflammatory axillary lymph nodes at nine months of age. Both had been vaccinated with BCG (a live attenuated strain of Mycobacterium bovis), which was injected into theshoulder a few days after birth. The enlarged lymph nodes were removed surgically. Biopsy confirmed the presence of acid-fast bacilli, consistent with mycobacterial infection. Neither of the patients had presented any other episode of mycobacterial infection by the time of clinical evaluation at three years of age.Ethics StatementThis study was conducted in accordance with the Helsinki Declaration, with written informed consent obtained from the parents of P1 and P2 and the other healthy individuals involved. Approval for this study was obtained from the French IRB (Comite ?de protection des personnes or CPP), INSERM and the Rockefeller IRB.Epstein-Barr Virus (EBV) Transformation of B Lymphocytes (EBV-B Cells) and Cell CulturePBMC were isolated from 10 ml of peripheral blood on a Ficoll gradient and were suspended in 4 ml of RPMI1640 supplemented with 20 fetal calf serum (FCS) and 0.2 mg/ml cyclosporine A. We then added 1 ml of EBV medium from the B95.8 cell line [28]. We replaced half the.
