Approval for this study was obtained from The University of British Columbia and Child & Family Research Institute. The research followed Canada's Tri-Council Statement on 'Ethical Conduct for Research Involving Humans'. Approval was also obtained through the Institutional Review Board of Self Regional Healthcare (Greenwood, SC). Consent from all patients was obtained for research purposes. Patients were ascertained and examined from five centers: The University of the Witwaterstrand, Johannesburg; The University of Cape Town, South Africa; Cedars-Sinai Medical Center, Los Angeles; Universita Cattolica, Rome; and The University of California, San Francisco. Patients were referred to the Greenwood Genetic Center for molecular research studies on split-hand/foot malformation. Controls were ascertained from the Greenwood Genetic Center.
All cases were sporadic and born to non-consanguineous parents. Patient 1 is the original patient with MMEP and t(6;13)(q21;q12) described previously by Viljoen and Smart . This is a 44-year-old Caucasian female with severe mental retardation, congenital microphthalmia causing complete blindness, central cleft lip and palate, ectrodactyly with absence of toes 2–4 on both feet, finger-like thumbs, and a broad, prominent jaw.
Patients 2–4 had normal blood chromosome analyses and were included in this study on the basis of having clinical features that significantly overlapped those of the MMEP phenotype. Patient 2 was felt to have possible MMEP versus an unusual variant of the ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome and has not been described previously. She is a Hispanic female with congenital microcephaly, bilateral microphthalmia, colobomas of the right iris and retina, left chorioretinal coloboma, left lacrimal duct stenosis, unilateral cleft lip and palate, dysplastic ears, soft tissue syndactyly of fingers 3–4 on the right hand, ectrodactyly of the left foot with a hallux and two digital rays, unilateral mixed versus sensorineural hearing loss requiring a hearing aid, sparse scalp hair and eyebrows, and narrow, deep-set nails. She had a prolonged hospitalization after birth and required gastrostomy tube placement. She had no evidence of a TP73L (previously, TP63, P63) mutation, which is known to be involved in split hand/foot malformation .
Patients 3 and 4 were felt to have an unusual variant of the EEC syndrome. A brief summary of their clinical findings was reported previously . Patient 3 is a 10-year-old Caucasian female with microcephaly, bilateral iris colobomas, microphthalmia with significant vision impairment, bilateral ectrodactyly of the hands and feet, unilateral cleft lip, and patchy alopecia of the scalp hair. She required gastrostomy feedings until age 9 due to ongoing problems with feeding, failure to thrive, and severe gastroesophageal reflux. A gastric emptying study revealed delayed emptying with a non-functioning section of the stomach and reverse peristalsis, which resolved with gastric Botox therapy. She has experienced significant dental problems due to ectodermal dysplasia. She attends a regular school program with visual assistance. Patient 4 is an African-American female seen at 3 months of age with congenital microcephaly, congenitally sealed eyelids with small to absent globes, ectrodactyly of the hands and feet, a notch in the upper lip resembling a mild midline cleft, absent scalp hair, underdeveloped eyelashes and eyebrows, underdeveloped nails, minor differences in ear shape, unilateral hearing loss, pelvic kidney, anteriorly placed anus, and tethered spinal cord. Both patients had no evidence of a TP73L mutation .
Patient 5 was included in the study on the basis of having a de novo chromosome translocation involving the 6q21 region (t(6;7)(q21;q31.2)) and congenital ulnar ray aplasia. His findings were described previously by Gurrieri et al. . He is a Caucasian male evaluated in the newborn period with congenitally bowed radii, absent ulnae, absence of fingers 3–5 on the right hand, syndactyly of fingers 2–3 and absence of fingers 4–5 on the left hand, and a cyst of the septum pellucidum. He did not have microcephaly, ocular abnormalities, or other features of the MMEP phenotype. We cannot exclude the possibility that breakage at 7q31 may disrupt a gene(s) involved in MMEP.
DNA amplification and sequencing
Sequencing was used to screen for SNX3 coding mutations in all 4 exons as previously described . We sequenced genomic NR2E1 using 20 PCR amplicons that covered the coding regions (1,146 bp), complete 5' and 3' UTRs (1,973 bp), exon-flanking regions including consensus splice-sites (1,719 bp), and evolutionarily conserved regions including the core and proximal promoter (1,528 bp). Polymerase chain reactions (PCR) and sequencing were performed as previously described . Sequences were visually inspected and scored blindly by at least two individuals using either Consed  or Sequencher (Gene Codes, Ann Arbor, MI). Every variant identified was confirmed by repeating the PCR and sequencing process. Confirmation of the g.21502T>C change in patient 2 and genotyping of unaffected parents and 250 controls without MMEP or related phenotypes was performed by restriction enzyme digestion of a 505-base pair PCR product using BsmBI. Primers and PCR conditions were as previously reported for 3' UTRb . The alteration created an additional BsmBI site, producing bands of 505, 278, and 227 base pairs in the heterozygous state.